Compositions and methods for treating ophthalmic infections and diseases

Abstract

To provide compositions and methods useful for detecting and diagnosing dry eye, treating dry eye, and developing treatment strategies and plans based on the diagnosis of dry eye.SOLUTION: The invention provides compositions and methods for identifying a subject suffering from dry eye that can be treated by topical administration of a composition comprising lacritin or a biologically-active fragment thereof. The present application discloses, in part, that an approximately 90kDa deglycanated form of syndecan-1 is abundant in tear fluid of normal individuals but not in individuals suffering from dry eyes, while an approximately 25kDa syndecan-1 fragment can be detected in dry but not normal tear fluid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT) This invention was made with United States government support under Grant Nos. RO1EY013143 and R01EY018222 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 61 / 951,680, filed March 12, 2014, and U.S. Provisional Application No. 62 / 019,476, filed June 1, 2014, the disclosures of each of which are incorporated herein by reference in their entireties.

[0003] (Incorporation by reference of electronically submitted materials) Incorporated by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing identified by a 19 kilobyte ACII (Text) file entitled "233965SeqListing.txt," filed concurrently herewith and created on May 11, 2015. [Background technology]

[0004] The health of the ocular surface depends on tear secretion from the lacrimal gland. The lacrimal acinar cells that comprise the lacrimal gland are polarized, well-differentiated lacrimal secretory cells attached to a complex pericinar basement membrane. Most of the apical cell cytoplasm contains large secretory granules packed with tear proteins. Known tear proteins include lysozyme, which plays a prominent bactericidal role at the corneal surface; lactoferrin, which functions as a bactericide and a potential inhibitor of complement activation; secretory components that control the intracellular movement of IgA into the acinar cavity while acting on the corneal surface to inhibit bacterial adhesion; and tear lipocalin (tear-specific prealbumin) and growth factors of unknown function, TGFα, TGFβ, and EGF. In rats, peroxidase is a tear component that serves as a convenient marker in experimental studies. Tears not only play an important bactericidal role, but also keep the cornea clean and lubricated, which is crucial for the health of the corneal epithelium.

[0005] The ocular surface is one of the most accessible and vulnerable tissues. Corneal epithelial cells face constant environmental insults, including UV radiation, widely varying temperature fluctuations, pollutants, bacteria, and other microbial organisms. Unlike other tissues, where blood vessels supply such agents, the cornea lacks a blood supply, making tears, which lubricate the corneal surface, the most promising source of cytoprotective and anti-inflammatory agents. In fact, tears are rich in antiseptic proteins. Dry eye patients suffering from insufficient tear production are prone to corneal ulceration, infection, or inflammation. Similar symptoms can occur with prolonged contact lens use due to the limited tear supply.

[0006] When tear production by lacrimal acinar cells is collectively insufficient, "dry eye" (also known as keratoconjunctivitis sicca [KCS]) results. Dry eye is a common ocular symptom of Sjögren's syndrome, an autoimmune disease of unknown etiology that affects millions of people worldwide. The most common affected individuals are postmenopausal women, with varying degrees of severity. If untreated, dry eye can lead to corneal abrasions, ulceration, bacterial infections, and vision loss. The molecular mechanisms responsible for the pathogenic reduction in secretion by the main lacrimal gland are potentially complex. The lacrimal glands of subjects with Sjögren's syndrome contain foci of B and T lymphocytes, the pathogenic expansion of which, potentially due to viral injury, can destroy the lacrimal acini. However, the loss of lacrimal gland volume sometimes appears insufficient compared with the theoretical excess capacity of the main lacrimal gland. Estimates suggest that the potential secretion volume is more than 10 times greater than that required to maintain a normal aqueous tear film layer. Therefore, other mechanisms, such as abnormal secretion of one or more common cytokines, that may directly or indirectly alter lacrimal acinar cell function and / or result in decreased innervation, merit attention. Novel autocrine / paracrine factors released by lacrimal acinar cells into the tear film may be required for the health of the lacrimal gland secretory system, ductal system, and corneal epithelium. The periacinar basement membrane is also required for normal secretory function, in part through "BM180," which apparently synergizes with laminin-1 to promote stimulated tear secretion. Alterations in each of these factors, either concomitant with or independent of normal changes, may contribute to decreased secretory capacity.

[0007] The lacrimal-corneal axis is a fundamental regulator of ocular health and plays a key role in ocular surface inflammation associated with dry eye syndrome and corneal injury. Numerous mediators, including the proinflammatory cytokines TNF-α, IL-1β, and IL-6, and the chemokine IL-8, are involved in the development and progression of corneal inflammation. Arachidonic acid-derived eicosanoids, generated by the activity of cyclooxygenases (primarily PGE2), lipoxygenases (12(s)-HETE), and cytochrome P450 (12(r)-HETE), also play a role.

[0008] Lacritin is a 12.3 kDa secreted glycoprotein released apically from human lacrimal acinar cells during reflex tearing and can be detected by ELISA and Western blot in mixed reflex and basal human tears. Lacritin is also produced by the epithelia of the cornea, conjunctiva, meibomian gland, and salivary gland as one of the most abundant eye-restricted genes. Recent studies on lacritin's mechanism of action show convergence with the PKCα and NFkB signaling pathways, suggesting that lacritin may have an important anti-inflammatory role on the ocular surface. Recent clinical studies support this hypothesis. A comparison of tear proteins from 19 subjects with blepharitis (eyelid inflammation) to 27 healthy volunteers revealed a 56% reduction in lacritin in the subjects. Sumadre et al. (2013) demonstrated that lacritin acutely increased basal tearing by 30% relative to vehicle and was well tolerated at multiple daily doses. It has also been reported that lacritin is selectively downregulated over other tear proteins in contact lens-associated dry eye. Lacritin stimulates MUC16 production by human corneal epithelial cells at levels that match or exceed those of serum (Non-Patent Document 2). Autologous serum has been reported to be a successful method for treating dry eye. Lacritin also promotes basal tear secretion by cultured rat and monkey lacrimal acinar cells and promotes the growth of human corneal epithelial cells.

[0009] Few cell types appear to be targetable by lacritin. Target cells include lacrimal acinar, salivary duct / HeLa, human corneal, and fetal kidney cells, but not others, among 17 different cell lines tested. Its coreceptor, syndecan-1, is widely expressed in the ocular surface epithelium. Thus, lacritin appears to be a multifunctional, eye-specific factor with potential roles in tear secretion and corneal epithelial regeneration.

[0010] There is a long-felt need in the art for compositions and methods useful for detecting and diagnosing dry eye, treating dry eye, and developing treatment strategies and regimens based on a diagnosis of dry eye. The present invention fulfills these needs. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Invest Ophthalmol Vis Sci., 2011;52:6265-6270; DOI:10.1167 / iovs.10-6220 [Non-patent document 2] Laurie GE, et al. IOVS 2006;47:ARVO E-Abstract 1606 Summary of the Invention

[0012] The present invention combines a novel mechanism for the molecular identification of dry eye disease with restorative therapies that address the cause. The invention relates to the discovery described herein that approximately 90 kDa deglycanized syndecan-1 is abundant in the tears of normal individuals but not in those suffering from dry eye. The 25 kDa syndecan-1 fragment is detectable in dry but not normal tears. The invention also relates to the discovery that topical lacritin, an agonist of deglycanized syndecan-1, sensitizes corneal sensory nerves to ocular surface dryness and increases the nerve's wet response. Thus, one embodiment of the invention relates to identifying dry eye by a relative decrease in approximately 90 kDa deglycanized syndecan-1 and / or the presence of 25 kDa syndecan-1 in tears. Another embodiment relates to increasing the dryness and wetness responses of corneal nerves by topical application of lacritin polypeptides to the eye.

[0013] The present applicant has also discovered that tears in aqueous-deficient dry eye are associated with a decrease in lacritin monomer, an increase in lacritin-C splice variant, and latent (chronically active) heparanase (HPSE). Thus, in one embodiment, a method is provided for identifying patients suffering from dry eye and selecting the patients for treatment. In one embodiment, a tear sample obtained from a subject contains: latent heparanase; 90kDa deglycanated SDC-1; 25kDa SDC-1; and inactive lacritin-C splice variant; The present invention provides a method for identifying a subject with dry eye, comprising detecting the presence of at least one protein selected from the group consisting of: a decrease in the level of active heparanase and a corresponding increase in active heparanase compared to the levels present in tears from normal eyes; a decrease in the levels of 90 kDa deglycanated SDC-1 compared with the levels present in tears from normal eyes; the presence of 25 kDa SDC-1; and / or Presence of an inactive lacritin-C splice variant The identified subject can then be treated by contacting the ocular surface of the subject's eye with a composition comprising lacritin or a bioactive fragment thereof.

[0014] Detection of latent heparanase, 90 kDa deglycanated SDC-1A, 25 kDa SDC-1, or inactive lacritin-C splice variants can be performed using standard techniques known to those skilled in the art, including the use of antibodies. In one embodiment, the antibodies can be embedded in Schirmer test strips onto which tears are collected for accurate and inexpensive molecular diagnosis in an ophthalmologist's or optometrist's office. Current methods for identifying subjects suffering from dry eye do not address the cause and are therefore plagued by inaccuracies and nonspecificity. Examples of current methods include: a) subject questionnaires, b) rose bengal or lissamine green staining of ocular surface damage, c) Schirmer test strip measurement of tear volume, d) tear breakup time, e) tear evaporation rate, f) tear meniscus height or half-system, g) tear film index or turnover rate, h) tear osmolality, i) lysozyme or lactoferrin assay, and j) tear fern analysis.

[0015] The return of active lacritin to the ocular surface has been shown to support the normal corneal sensory nerve dryness and moistening responses necessary for physiologically normal eyes. Because all glands that moisten the eye are controlled by reflex arcs downstream of corneal sensory input, lacritin or lacritin fragments, synthetic peptides, or mimetics should benefit all forms of dry eye. Preclinical studies in rabbit and dry eye mouse models suggest that the density of corneal sensory innervation, which is reduced in dry eye, may be restored. In contrast, commonly used "artificial tears" temporarily relieve symptoms without addressing the cause.

[0016] The present disclosure relates to the tear fluid of aqueous-deficient dry eye, which is associated with a decrease in lacritin monomer, an increase in lacritin-C splice variants, less deglycanized SDC1, an increase in the 25 kDa SDC1 fragment, and a decrease in latent heparanase and an increase in active heparanase. Accordingly, the present invention provides compositions and methods for detecting and diagnosing dry eye, and for developing and providing treatment plans for subjects found to have dry eye using one or more of the dry eye markers described herein. The present application also provides compositions and methods for detecting and diagnosing dry eye, including the FOXO3 translocation assay described herein. Methods are also available and described for detecting and measuring proteins and protein fragments useful for detecting and diagnosing dry eye.

[0017] The present invention further provides the use of lacritin, or a biologically active fragment or analog thereof, on corneal sensory nerves to treat dryness of the ocular surface, increasing the moistening response of the nerves. In one embodiment, topical use of lacritin or fragment N-94 (SEQ ID NO: 7) restores or increases tearing. In one embodiment, the use restores basal tearing. In one embodiment, topical administration of lacritin suppresses lacrimal gland inflammation.

[0018] According to one embodiment, a treatment method is provided for restoring levels of 90 kDa syndecan-1 (SDC1) or other deglycanated forms of syndecan-1 in the tears of a subject with dry eye. Restoration of SDC1 enhances the activity of existing lacritin. The present invention further provides a method for treating dry eye using inhibitors of transglutaminase (TGM), which can be inhibitors of TGM activity, levels, or synthesis.

[0019] According to one embodiment, there is provided a composition comprising a peptide, non-naturally occurring peptide, or peptidomimetic derivative comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or a sequence which differs from SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 by 1, 2, 3, 4, or 5 amino acids, or a biologically active fragment, homolog, or derivative thereof. In one embodiment, the peptide differs from SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:7 by 1, 2, 3, 4, or 5 conservative amino acid substitutions. In one embodiment, the amino acid modification is an amino acid substitution, and in one embodiment, the substitution is a conservative amino acid substitution.

[0020] In some embodiments, a peptide of the present disclosure comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:7, or a biologically active fragment, homolog, or derivative thereof.

[0021] In some embodiments, peptides of the present disclosure comprise a non-natural amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 7, or a peptidomimetic derivative of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 7. The statement that a peptide is non-natural is intended to exclude the naturally occurring peptide of the parent lacritin peptide.

[0022] According to one embodiment, there is provided a method of enhancing corneal wound healing in a subject in need thereof. The method comprises contacting the ocular surface of the subject with a composition comprising lacritin or a bioactive fragment thereof. In one embodiment, the bioactive fragment of lacritin is KQFIENGSEFAQKLLKKFS(SEQ ID NO:5); KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7); KQFIENGSEFANKLLKKFS (SEQ ID NO: 6); and KQFIENGSEFANKLLKKFSLLKPWA (SEQ ID NO: 8), or a derivative thereof that differs from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 by one or two amino acid substitutions. In one embodiment, the subject has recovered from PRK (laser-assisted refractive keratectomy) or LASIK (laser-assisted in situ keratomileusis) surgery.

[0023] In another embodiment, a bactericidal composition is provided comprising a C-terminal fragment of lacritin. In one embodiment, the fragment is a peptide selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or a derivative thereof that differs from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 by one or two amino acid substitutions. In one embodiment, the fragment is a peptide consisting of the sequence of SEQ ID NO:7. In one embodiment, the composition comprises a pharmaceutically acceptable carrier, wherein the composition is suitable for topical administration to the ocular surface of a subject. In one embodiment, the composition further comprises a second antibacterial agent. As described herein, there is provided a method of treating a corneal infection, wherein the method comprises contacting the cornea of ​​a subject in need thereof with a composition comprising a C-terminal fragment of lacritin. [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1 shows the detection of dilute deglycanized and 25 kDa forms of syndecan-1 in normal and dry eye tears, respectively. In the paired specimens on the left (before laser photorefractive keratectomy (PRK) or laser in situ ablation keratomileusis (LASIK)), approximately 90 kDa deglycanized SDC1 is abundant in normal tears and barely detectable in dry eye tears. One day after PRK or LASIK surgery (D1), approximately 90 kDa deglycanized SDC1 is less abundant in dry eye tears. The 25 kDa SDC1 fragment is also evident in dry eye tears. Surgery promotes dry eye by severing corneal sensory nerves. One week after PRK or LASIK surgery (W1), approximately 90 deglycanated SDC1 levels are restored in normal operated tears, and 25 kDa levels remain elevated in dry eye tears (less so at 1 month (M1)). [Figure 2] Figures 2A and 2B show the detection of elevated levels of the inactive lacritin-C splice variant in tears of dry eye. Figure 2A is a Western blot showing the detection of lacritin-C using mab 4F6. The lacritin-C splice variant is consistently elevated in dry eye. Figure 2B is a Western blot using the secondary antibody alone, which serves as a negative control. No band is detected using the secondary antibody alone. [Figure 3] Figures 3A and 3B show the detection of more latent heparanase in normal versus dry eye tears, and more active heparanase in dry eye tears. Figure 3A is a Western blot showing heparanase using antibody #1453. Latent heparanase is indicated by an approximately 75 kDa band; active heparanase is indicated by an approximately 50 kDa band. Figure 3B is a Western blot showing heparanase using antibody #753. [Figure 4]Figures 4A-4C show the corneal health-restoring activity of lacritin and the C-terminal 25-amino acid fragment of lacritin (LACRIPEP). Cultured human corneal epithelial cells were treated with inflammatory cytokines to induce stress, and the cells were then treated with 10 nM of an inactive lacritin truncation mutant (C-25), lacritin, or LACRIPEP. Measurement of cytoplasmic staining in a FOXO3 assay (nuclear FOXO3 staining indicates cell death) indicates that LACRIPEP is equally active as lacritin (see Figure 4A) in enhancing cell survival compared to the negative control (C-25). Studies in dry eye (Aire- / -) mice also demonstrate the bioactivity of topically administered LACRIPEP. LACRIPEP prevents tear loss and causes dry eye disease in Aire(- / -) dry eye mice (Figure 4B; filled circles) compared to those given topical PBS (open circles), and Aire(- / -) dry eye mice given LACRIPEP had less corneal staining, indicating cell death, because LACRIPEP prevents tear loss and causes dry eye disease compared to those given topical PBS (open circles) (Figure 4C; filled circles). [Figure 5] Figure 5 shows the comparative survival-promoting activity of lacritin and synthetic lacritin peptides. Quantification of FOXO3 immunostaining in interferon-γ and tumor necrosis factor-positive cells in stressed human HCE-T cells treated with lacritin C-terminal truncation mutant C-25 (negative control; inactive), lacritin (lacrt), lacritin C-terminal peptides N-94 (SEQ ID NO: 7) or N-94 / C-6 (SEQ ID NO: 5), or tissue transglutaminase-polymerized lacritin (inactive). The dose of each peptide administered was 10 nM. Increased nuclear staining indicates stress / death. Increased cytoplasmic staining (arrows) indicates survival. Between 203 and 379 cells were counted for each treatment. Comparisons of all but polymerized lacrt vs. C-25 were performed by two-way ANOVA with Bonferroni posttest, P = 0.01. [Figure 6]Figures 6A and 6B show that LACRIPEP exhibits surprising stability in human tears. Figure 6A shows immunoblots of the protease-sensitive positive control "SN pep" and LACRIPEP ("N-94"; SEQ ID NO: 7) from various proteins after incubation in lacritin-depleted human tears at 37°C for 2 to 16 hours. Figure 6B shows mass spectrometry analysis, where the top panel shows MS profiles of SN pep, LACRIPEP ("N-94"), and LACRIPEP lacking the six C-terminal amino acids ("N-94 / C-6") before the addition of tear fluid and the 37°C incubation step, and the bottom panel provides MS profiles after incubation in lacritin-depleted tears for 4 hours at 37°C. [Figure 7] Figures 7A and 7B show the biphasic dose response of topical LACRIPEP in a rabbit basal tearing test (Figure 7A) and in a rat corneal sensory nerve stimulation (pLAC; Figure 7B) test compared to an inactive lacritin fragment control (C-25D). [Figure 8] Figure 8 shows the distribution of a single 4 μM dose of topical 125I-Lacripep-Y on the eye of a rat. Small amounts of 125I-Lacripep-Y are detectable in the blood and plasma. A significant amount is retained in the tears. [Figure 9] Figure 9 shows an alignment of the 25 amino acid C-terminal fragments of lacritin homologs from primate species, including human (SEQ ID NO: 7); chimpanzee (SEQ ID NO: 17); bush baby (SEQ ID NO: 18); gorilla (SEQ ID NO: 19); macaque (SEQ ID NO: 20); marmoset (SEQ ID NO: 21); mouse lemur (SEQ ID NO: 22) and orangutan (SEQ ID NO: 23), demonstrating high sequence conservation among primate species. DETAILED DESCRIPTION OF THE INVENTION

[0025] Abbreviations and Acronyms FACS stands for fluorescence activated cell sorter. HCE means human corneal epithelium. HPSE means heparanase. HS means heparin sulfate. HSG stands for human salivary gland. INFG stands for interferon gamma (also indicated as IFNG). IRB stands for Institutional Review Board. SDC1 means syndecan-1. TGM means transglutaminase. TNF means tumor necrosis factor.

[0026] (definition) In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0027] As used herein, the term "lacritin polypeptide" and like terms are defined as any peptide comprising the amino acid sequence of SEQ ID NO: 1 and / or a biologically active fragment, homolog, or derivative thereof. As used herein, the term "biologically active fragment" or "bioactive fragment" of a lacritin polypeptide encompasses naturally occurring or synthetic portions of the amino acid sequence MKFTTLLFLAAVAGALVYAEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 1). Fragments of lacritin (SEQ ID NO: 1) include, for example: KQFIENGSEFAQKLLKKFS (SEQ ID NO: 5) ("N-94 / C-6") (Wang et al., (2006) J. Cell Biol. 174, 689-700), and KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7) ("N-94") (see Zhang et al., (2013) J. Biol. Chem. 288, 12090-12101).

[0028] As used herein, the term "about" means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical value or range, it modifies that range by expanding the boundaries above and below the stated numerical values. For example, in one embodiment, the term "about" is used to modify a numerical value above and below the indicated value by a variance of 20%, without intending to assign any value or range of values ​​to this broader definition. Each value or range of values ​​preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.

[0029] As used herein, an "acylated" amino acid is an amino acid that contains an acyl group that is unnatural to natural amino acids, regardless of how it is produced. Typical methods for producing acylated amino acids and acylated peptides are known in the art and include acylation of the amino acid prior to incorporation into the peptide or chemical acylation of the peptide after peptide synthesis. In some embodiments, the acyl group confers one or more of the following benefits on the peptide: (i) increased half-life in the blood circulation; (ii) delayed onset of action; (iii) prolonged duration of action; and (iv) increased resistance to proteases.

[0030] As used herein, an "alkylated" amino acid is an amino acid that contains an alkyl group that is unnatural to natural amino acids, regardless of the method by which it is produced. Typical methods for producing alkylated amino acids and alkylated peptides are known in the art and include alkylating the amino acid before incorporation into the peptide or chemically alkylating the peptide after peptide synthesis. Without being bound by any particular theory, it is believed that alkylating a peptide will achieve similar, if not identical, effects to acylation of a peptide, such as prolonged half-life in blood circulation, delayed onset of action, prolonged duration of action, and improved resistance to proteases.

[0031] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents. The term also encompasses any substance approved by a regulatory agency of the United States Federal government for use in animals, including humans, or listed in the US Pharmacopoeia.

[0032] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that retains the biological activity of the parent compound and which is not biologically or otherwise undesirable. Many of the compounds described herein are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0033] As used herein, the term "hydrophilic moiety" refers to any compound that is readily soluble in or absorbs water and can be tolerated in vivo by mammalian species without toxic effects (i.e., is biocompatible). Examples of hydrophilic moieties include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polylactic-polyglycolic acid copolymers, polyvinyl alcohol, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyl methacrylate, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and cellulose derivatives such as hydroxymethylcellulose or hydroxyethylcellulose and their copolymers, as well as natural polymers including, for example, albumin, heparin, and dextran.

[0034] An experimental, diagnostic, or treatment "subject" is an animal, including a human.

[0035] As used herein, the term "dry eye" encompasses any condition in which there is insufficient tear fluid to moisturize and nourish the eye. Subjects with dry eye do not produce enough tears or have poor tear quality. As used herein, dry eye includes, but is not limited to, aqueous-deficient and evaporative dry eye. Aqueous-deficient dry eye includes, but is not limited to, Sjogren's syndrome dry eye (including primary and secondary), non-Sjogren's dry eye (including those caused by tear deficiency, lacrimal duct obstruction, reflex blockage, and systemic medications). Evaporative dry eye includes, but is not limited to, intrinsic (including those caused by meibomian oil deficiency, palpebral fissure obstruction, low blink rate, and Accutane medication) and extrinsic (including vitamin A deficiency, topical medication preservatives, contact lens wear, and ocular surface disease (e.g., allergies)).

[0036] As used herein, the term "treating" includes preventing a particular disorder or condition, alleviating symptoms associated with a particular disorder or condition, and / or preventing or eliminating said symptoms. For example, as used herein, the term "treating dry eye" generally refers to maintaining basal tear levels near normal levels, and may include increasing tear levels depending on the given situation.

[0037] As used herein, the term "effective" or "therapeutically effective" amount of a pharmaceutical agent refers to a non-toxic but sufficient amount of the agent to provide a desired effect. For example, one desired effect may be the prevention or treatment of dry eye. The amount that is "effective" will vary from subject to subject, depending on age, individual's general condition, mode of administration, etc. Therefore, it is not always possible to specify an exact "effective amount." However, the appropriate "effective" amount in any individual case can be determined by those skilled in the art through routine testing.

[0038] The term "additional therapeutically active compound" or "additional therapeutic agent" as used in connection with the present invention refers to the use or administration of a compound for additional therapeutic use for the particular injury, disease, or disorder being treated. For example, such compounds could include unrelated diseases or disorders, or diseases or disorders that may not respond to initial treatment for the injury, disease, or disorder being treated.

[0039] As used herein, the term "identity" refers to the similarity between two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the result by 100 to arrive at a percentage. Thus, two copies of the exact same sequence will have 100% identity, while two sequences that have amino acid / nucleic acid deletions, additions, or substitutions compared to each other will have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity, such as those that use algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215:403-410).

[0040] As used herein, an amino acid "modification" refers to the substitution of an amino acid or the derivation of an amino acid by the addition and / or removal of chemical groups to / from an amino acid, and includes substitution with either the 20 amino acids commonly found in human proteins, as well as variant or unnatural amino acids. Commercial suppliers of variant amino acids include Sigma-Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA). Variant amino acids can be purchased from commercial suppliers, synthesized de novo, or chemically modified or derived from natural amino acids.

[0041] As used herein, an amino acid "substitution" refers to the replacement of one amino acid residue with a different amino acid residue.

[0042] As used herein, the term "conservative amino acid substitution" is defined herein as an exchange within one of the following five groups: I. Small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar negatively charged residues and their amides: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; III. Polar positively charged residues: His, Arg, Lys; Ornithine (Orn) IV. Large aliphatic non-polar residues: Met, Leu, Ile, Val, Cys, Nor-leucine (Nle), homocysteine V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine

[0043] As used herein, the term "isolated" means removed from its natural environment. In some embodiments, the peptide is made recombinantly and the peptide is isolated from a host cell.

[0044] The term "purified," as defined herein, refers to the isolation of a molecule or compound in a form that is essentially free from contaminants normally associated with the molecule or compound in its native or natural environment, and refers to increased purity as a result of being separated from other components of the original composition. The term "purified polypeptide" is used herein to describe a polypeptide that has been separated from other compounds, including, but not limited to, nucleic acid molecules, lipids, and carbohydrates.

[0045] "Peptide mimetic" refers to a chemical compound having a structure that differs from the general structure of an existing peptide, but functions similarly to an existing peptide, for example, by mimicking the biological activity of that peptide. Peptide mimetics typically contain natural and / or unnatural amino acids, but can also include modifications to the peptide backbone. For example, a peptidomimetic can include a natural amino acid sequence with the insertion or substitution of a non-peptide moiety, such as a retro-inverso fragment, or the incorporation of a non-peptide bond, such as an azapeptide bond (CO replaced by NH), or a pseudopeptide bond (e.g., NH replaced by CH), or an ester bond (e.g., a depsipeptide in which one or more of the amide (-CONHR-) bonds are replaced by an ester (COOR) bond). Alternatively, a peptidomimetic can lack natural amino acids altogether.

[0046] As used herein, the terms "administration of" and / or "administering a" compound should be understood to mean providing a compound of the present invention to a subject in need of treatment.

[0047] As used herein, amino acids are represented by their full name, their corresponding three-letter code, or their corresponding one-letter code, as shown in the table below. [Table 1]

[0048] As used herein, the term "amino acid" encompasses any molecule containing both amino and carboxyl functional groups, where the amino and carboxylate groups are attached to the same carbon (the α-carbon). The α-carbon may optionally bear one or two additional organic substituents. For purposes of this disclosure, the designation of an amino acid without specification of stereochemistry is intended to encompass either the L- or D-form of the amino acid, or a racemic mixture. However, when an amino acid is designated by its three-letter code and includes a superscript number, the D-form of the amino acid is specified by including a lowercase d and a superscript number before the three-letter code (e.g., dLys1), where the designation lacking the lowercase d (e.g., Lys1) is intended to indicate the native L-form of the amino acid. In this nomenclature, the inclusion of a superscript number designates the position of the amino acid in a peptide sequence numbered consecutively from the N-terminus. As used herein, the expression "amino acid" is intended to include both natural and synthetic amino acids, and both D- and L-form amino acids. A "standard amino acid" refers to any of the 20 L-amino acids commonly found in naturally occurring peptides.

[0049] As used herein, the term "non-encoded amino acid" includes any amino acid that is not the L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, whether it is synthetically prepared or derived from a natural source.

[0050] A general reference to a peptide as used herein is intended to encompass peptides with modified amino and carboxy termini, including, but not limited to, salts. For example, an amino acid sequence specifying a standard amino acid is intended to encompass the standard amino acid at the N- and C-termini, and the corresponding hydroxyl acid at the N-termini and / or the corresponding modified C-terminal amino acid, containing an amide group at the terminal carboxylic acid position. Amino acids contained within the peptides of the present invention, particularly those at the carboxy or amino termini, can be modified by methylation, amidation, acetylation, or substitution with other chemical groups, which can alter the circulating half-life of the peptides without adversely affecting their activity. Furthermore, disulfide bonds may or may not be present in the peptides of the present invention.

[0051] The term "amino acid" is used interchangeably with "amino acid residue" and can refer to free amino acids and amino acid residues of peptides. Whether a free amino acid or a residue of a peptide is meant, it will be understood from the context which term is used.

[0052] Amino acids can be classified into seven groups based on the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxyl (OH) group, (3) side chains containing a sulfur atom, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline (an imino acid with a side chain fused to an amino group).

[0053] The nomenclature used to describe the peptide compounds of the invention follows conventional practice, with the amino group presented to the left and the carboxy group presented to the right of each amino acid residue. In formulae representing selected specific embodiments of the invention, the amino and carboxy terminal groups are not specifically shown, but will be understood to be in the form they appear at physiological pH values ​​unless otherwise specified.

[0054] As used herein, the term "basic" or "positively charged" amino acid refers to an amino acid in which the R group carries a net positive charge at pH 7.0, including, but not limited to, the standard amino acids lysine, arginine, and histidine.

[0055] As used herein, the term "antibody" refers to an antibody that specifically binds to a specific epitope on an antigen. "Antibody" refers to an immunoglobulin molecule capable of binding to a target molecule. An antibody can be an intact immunoglobulin derived from a natural source or a recombinant source, or can be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The antibodies of the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0056] As used herein, "antimicrobial agent" means any compound that prevents the growth of or kills any microorganism.

[0057] As used herein, "bactericide" means any compound that prevents the growth of or kills any bacteria.

[0058] As used herein, the term "biologically active fragment" or "bioactive fragment" of a polypeptide encompasses natural or synthetic portions of full-length proteins that are capable of specifically binding to their natural ligands or performing the functions of the proteins.

[0059] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA."

[0060] Use of the word "detect" and its grammatical variations means the measurement of a species without quantification, while use of the words "determine" or "measure," along with their grammatical variations, means the measurement of a species with quantification. The terms "detect" and "identify" are used interchangeably herein.

[0061] As used herein, a "detectable marker" or "reporter molecule" is an atom or molecule that allows for the specific detection of a compound containing the marker in the presence of similar compounds without the marker. Detectable markers or reporter molecules include, for example, radioisotopes, antigenic determinants, enzymes, nucleic acids available for hybridization, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, and molecules that provide modified fluorescence polarization or modified light scattering.

[0062] As used herein, the phrase "enhancing survival" refers to reducing the amount or rate of death in a cell population. Enhancing survival can be by preventing cell death alone (e.g., cell death associated with apoptosis) or by reducing the rate of cell death. Reducing cell death can also result from indirect effects, such as inducing some cell proliferation, which effectively replenishes at least some or all of the cell population as cells die. Enhancing cell survival can also be achieved by a combination of inducing proliferation and reducing cell death or the rate of cell death. "Promoting survival" and "enhancing viability" are used interchangeably herein with "providing survival enhancement."

[0063] A "fragment" or "segment" is a portion of an amino acid sequence comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms "fragment" and "segment" are used interchangeably herein. Fragments of lacritin peptides used herein as part of compositions for use in treatment or to induce a lacritin effect are presumed to be biologically active fragments with respect to the response to be induced.

[0064] As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property or activity by which it is characterized. For example, a functional enzyme is an enzyme that exhibits the characteristic catalytic activity by which the enzyme is characterized.

[0065] As used herein, "gene" refers to a nucleic acid coding sequence and the regulatory elements necessary for the transcription of that DNA sequence into messenger RNA (mRNA) and subsequent translation into a sequence of amino acids characteristic of a specific polypeptide.

[0066] As used herein, the term "insult" refers to contact with a substance or environmental change that results in an alteration of normal cellular metabolism in a cell or population of cells. Environmental insults include chemicals, environmental pollution, heavy metals, viral or bacterial infections, changes in temperature, changes in pH, and acidity. These may include, but are not limited to, agents that cause damage, DNA damage, or pathogenicity. The term "damage" is used interchangeably herein with "environmental damage."

[0067] As used herein, the term "syndecan-1" refers to a peptide comprising the amino acid sequence of SEQ ID NO: 2, as well as biologically active fragments, derivatives, and homologs thereof. As used herein, the term "biologically active fragment" or "bioactive fragment" of a syndecan-1 polypeptide refers to a peptide comprising the amino acid sequence MRRAALWLWLCALALSLQPALP QIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQ ASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQKPTKQEEFYA (SEQ ID NO: 2). The underlined portion of SEQ ID NO: 2 represents the sequence: It represents the "shed deglycanized 90 kDa form of syndecan-1" (or 90 kDa deglycanized SDC-1), having the amino acid sequence QIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKE (SEQ ID NO: 3).

[0068] As used herein, the term "25 kDa fragment of SDC-1 ectodomain" (or 25 kDa SDC-1) refers to a 25 kDa fragment of SDC-1 that contains the LPEV sequence of 90 kDa deglycanized SDC-1.

[0069] As used herein, the term "heparanase" refers to a peptide comprising the amino acid sequence of SEQ ID NO: 4, as well as biologically active fragments, derivatives, and homologs thereof. As used herein, the term "biologically active fragment" or "bioactive fragment" of a heparanase polypeptide encompasses naturally occurring or synthetic portions of the amino acid sequence (SEQ ID NO: 4).

[0070] As used herein, a "ligand" is a compound that specifically binds to a target compound. A ligand (e.g., an antibody) "specifically binds" or is "specifically immunoreactive" with a compound if the ligand functions in a binding reaction that determines the presence of the compound in a sample of heterogeneous compounds. Thus, under specified assay (e.g., immunoassay) conditions, the ligand preferably binds to a specific compound and does not bind to a significant extent to other compounds present in the sample. For example, an antibody specifically binds to the antigen bearing the epitope to which the antibody was raised under immunoassay conditions. Various immunoassay formats may be used to select antibodies that specifically immunoreact with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immunoreact with an antigen. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0071] As used herein, the term "linkage" refers to a connection between two groups. The connection can be either a covalent bond or a non-covalent bond, including but not limited to ionic bonds, hydrogen bonds, and hydrophobic / hydrophilic interactions.

[0072] As used herein, the term "linker" means a molecule that connects two other molecules either covalently or non-covalently, for example, via ionic or hydrogen bonds or van der Waals interactions.

[0073] As used herein, "ocular surface" means the surface of the eye, particularly the surface of the cornea.

[0074] As used herein, the phrase "ocular surface disease, injury, or condition" means any disease, disorder, or condition that directly or indirectly causes or is capable of causing any of the problems or symptoms described herein for an ocular surface disease, disorder, or condition.

[0075] "Operably linked" refers to a juxtaposition wherein the components are configured to perform their function. Thus, a control sequence or promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence.

[0076] A "marker" is an atom or molecule that allows specific detection of a molecule containing the marker in the presence of similar molecules without the marker. Markers include, for example, radioisotopes, antigenic determinants, nucleic acids available for hybridization, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, molecules that provide modified fluorescence polarization or modified light scattering, and molecules that enable enhanced survival of cells or organisms (i.e., selectable markers). A reporter gene is a gene that encodes a marker.

[0077] As used herein, the terms "measuring expression levels" or "determining expression levels" refer to any measurement or assay that can be used to correlate the results of an assay with the expression level of a gene or protein of interest. Such assays include measurements of mRNA levels, protein levels, etc., and can be performed by assays such as Northern blot analysis, Western blot analysis, binding assays, immunoblots, etc. Expression levels can include expression rates and can be measured by the actual amount of mRNA or protein present. Such assays are associated with processes or systems to store and process information, to help quantify levels, signals, etc., and to digitize information for use in comparing levels.

[0078] A "polylinker" is a nucleic acid sequence that contains a series of three or more different restriction endonuclease recognition sequences that are closely spaced from each other (ie, less than 10 nucleotides between each site).

[0079] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence that is necessary for the expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements that are necessary for the expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

[0080] A "constitutive promoter" is a promoter that drives expression of a gene to which it is operably linked in a constant manner in a cell. For example, a promoter that drives expression of a cellular housekeeping gene is considered a constitutive promoter.

[0081] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a living cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0082] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0083] As used herein, "nucleic acid," "DNA," and similar terms also include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are also considered within the scope of the present invention.

[0084] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs may contain introns.

[0085] The term "peptide" encompasses a sequence of three or more amino acids, where the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids. Peptide mimetics include peptides with one or more of the following modifications: 1. Peptides in which one or more peptidyl --C(O)NR-- linkages (bonds) are replaced by a non-peptidyl linkage such as a --CH2-carbamate linkage (--CH2OC(O)NR--), a phosphonate linkage, a --CH2-sulfonamide (--CH2--S(O)2NR--), a urea (--NHC(O)NH--), a --CH2-secondary amine linkage, or an alkylated peptidyl linkage (--C(O)NR--), where R is C1-C4 alkyl; 2. Peptides derivatized at the N-terminus with a --NRR1, --NRC(O)R, --NRC(O)OR, --NRS(O)2R, or --NHC(O)NHR group, where R and R1 are hydrogen or C1-C4 alkyl, with the proviso that R and R1 are not both hydrogen; 3. Peptides whose C-termini are derivatized to --C(O)R2, where R2 is selected from the group consisting of C1-C4 alkoxy, and --NR3R4, where R3 and R4 are independently selected from the group consisting of hydrogen and C1-C4 alkyl.

[0086] Synthetic or non-naturally occurring amino acids refer to amino acids that do not naturally occur in vivo but that can nevertheless be incorporated into the peptide structures described herein. The resulting "synthetic peptides" contain amino acids other than the 20 naturally occurring, genetically encoded amino acids at one, two, or more positions in the peptide. For example, naphthylalanine can be substituted for tryptophan to facilitate synthesis. Other synthetic amino acids that can be substituted into peptides include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, α-amino acids such as L-α-hydroxylysyl and D-α-methylalanyl, L-α-methylalanyl, β-amino acids, and isoquinolyl. D-amino acids and non-naturally occurring synthetic amino acids can also be incorporated into peptides. Other derivatives include replacing the naturally occurring side chains of the 20 genetically encoded amino acids (or any L or D amino acid) with other side chains.

[0087] The term "fusion polypeptide" or "fusion protein" refers to a chimeric protein comprising a reference protein (e.g., lacritin) linked at its N- and / or C-terminus to one or more heterologous sequences (e.g., a non-lacritin polypeptide, such as a syndecan). Because the peptide linkage occurs between the backbone amino group of a first amino acid residue and the carboxyl group of a second amino acid residue, the polypeptide molecule is said to have an "amino terminus" (N-terminus) and a "carboxy terminus" (C-terminus). The terms "N-terminus" and "C-terminus" with respect to a polypeptide sequence refer to regions of the polypeptide that include portions of the N- and C-terminal regions of the polypeptide, respectively. Sequences that include portions of the N-terminal region of the polypeptide primarily, but are not limited to, amino acids from the N-terminal half of the polypeptide chain. For example, an N-terminal sequence can include polypeptide sequences that include bases from both the N- and C-terminal halves of the polypeptide. The same applies to the C-terminal region. The N- and C-terminal regions can, but do not necessarily, include amino acids that define the final N- and C-termini of the polypeptide, respectively.

[0088] The fusion proteins of the present invention can be produced by recombinant methods or solid-phase chemical peptide synthesis. This method has been known in the art since the early 1960s (Merrifield, 1963) (see also Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill., pp. 11-12) and is currently used in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Additionally, many FMOC peptide synthesis systems are available. For example, assembly of polypeptides or fragments can be performed on a solid support using an Applied Biosystems, Inc. Model 431A automated peptide synthesizer. This equipment provides ready access to the peptides of the present invention either by direct synthesis or by synthesis of a series of fragments that can be joined using other known techniques.

[0089] The present invention also includes stable cell lines expressing expression cassettes comprising a lacritin bioactive fragment or a lacritin / syndecan-1 fusion protein, a nucleic acid molecule encoding the lacritin fragment or the lacritin / syndecan-1 fusion protein, and a vector capable of expressing the nucleic acid molecule of the invention in a host cell. Preferably, the expression cassette comprises a promoter, e.g., a constitutive or regulatable promoter, operably linked to the nucleic acid sequence. In one embodiment, the expression cassette comprises an inducible promoter. Also provided are host cells, e.g., prokaryotic or eukaryotic cells, e.g., plants or vertebrate cells, e.g., mammalian cells (including, but not limited to, human, non-human primate, canine, feline, bovine, equine, ovine, or rodent (e.g., rabbit, rat, ferret, or mouse) cells), comprising the expression cassette or vector of the invention, as well as kits comprising the nucleic acid molecule, expression cassette, vector, host cell, or lacritin / syndecan-1 fusion protein.

[0090] The term "vector" also includes synthetic compounds that can be used to deliver isolated nucleic acids, including isolated nucleic acids, into cells. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, ionic or amphiphilic compounds, plasmids, and virus-related polynucleotides. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, plasmids, cosmids, lambda phage vectors, and the like.

[0091] "Expression vector" means a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses that incorporate recombinant polynucleotides.

[0092] As used herein, the term "wound" relates to a physical tear or disruption to a tissue or cell layer. A wound can be caused by any physical injury, including surgery.

[0093] Implementation Compositions having lacritin-based activity as described herein are disclosed for treating diseases, injuries, or conditions related to the ocular surface. According to one embodiment, a composition is provided comprising a lacritin polypeptide, a biologically active fragment of lacritin, a non-natural lacritin peptide, or a peptidomimetic derivative of lacritin. In one embodiment, the composition comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 5, and SEQ ID NO: 6, or a sequence that differs from SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 5, or SEQ ID NO: 6 by one, two, three, four, or five amino acid modifications. In one embodiment, the composition comprises a sequence selected from the group consisting of SEQ ID NO: 7 or SEQ ID NO: 8, or a sequence that differs from SEQ ID NO: 7 or SEQ ID NO: 8 by one, two, three, four, or five amino acid substitutions, and in a further embodiment, the one, two, three, four, or five amino acid substitutions are conservative amino acid substitutions.

[0094] In one embodiment, a composition is provided comprising a biologically active fragment of lacritin, wherein the biologically active fragment consists of the sequence of SEQ ID NO:7, or a derivative that differs from SEQ ID NO:7 by a single amino acid substitution. In one embodiment, the single amino acid substitution is a conservative amino acid substitution, and in a further embodiment, the amino acid substitution is at positions 4, 6, 8, 10, 17, and 19. In one embodiment, the biologically active fragment consists of the sequence of SEQ ID NO:7, or a derivative that differs from SEQ ID NO:7 by a single amino acid substitution at position 4 or 19. Surprisingly, Applicants have discovered that a 25-amino acid C-terminal fragment of native human lacritin (SEQ ID NO:7) has enhanced stability in human tears compared to the same fragment with the terminal 6 amino acids removed (SEQ ID NO:5). In particular, immunoblotting revealed that after incubation in lacritin-depleted tears at 37°C for 4 hours, N-94 / C-6, but not Lacripep ("N-94"; SEQ ID NO:7), loses its epitope.

[0095] Although topical application of ophthalmic products remains the most popular and well-tolerated route of administration due to patient compliance, the bioavailability of eye drops is severely hindered by blinking, basal and reflex tear secretion, and nasolacrimal drainage. One solution to enhance the therapeutic index of topical treatments is the application of polymeric nanoparticles as drug carriers. According to one embodiment, a pharmaceutical composition is provided comprising lacritin or a bioactive fragment thereof linked to nanoparticles. In one embodiment, the nanoparticles are thermoresponsive elastin-like polypeptides (ELPs). ELPs are composed of a repeating pentapeptide motif (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 24) and exhibit a unique reversible inverse phase transition temperature, Tt, below which they become soluble and above which they phase separate. In one embodiment, the carboxy terminus of lacritin or a bioactive fragment thereof is linked to an ELP. In one embodiment, the C-terminus of a peptide consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 is a repeating pentapeptide motif (VPGSG). 48 (VPGIG) 48 (SEQ ID NO: 28).

[0096] According to one embodiment, a composition is provided comprising a syndecan-1 peptide, a non-natural peptide, or a peptidomimetic derivative thereof. In one embodiment, the peptide comprises a sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3, or a sequence that differs from SEQ ID NO:2 and SEQ ID NO:3 by 1, 2, 3, 4, or 5 amino acids, and homologs and fragments thereof. In one embodiment, the peptide differs from SEQ ID NO:2 and SEQ ID NO:3 by 1, 2, 3, 4, or 5 conservative amino acid substitutions. In one embodiment, the amino acid modification is an amino acid substitution, and in one embodiment, the substitution is a conservative amino acid substitution. In one embodiment, the composition comprises a syndecan-1 fragment consisting of the sequence of SEQ ID NO:2.

[0097] In some embodiments, a peptide of the present disclosure comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3, or a fragment or homolog thereof.

[0098] In some embodiments, a peptide of the present disclosure comprises a non-naturally occurring amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a peptidomimetic derivative of SEQ ID NO: 2 or SEQ ID NO: 3. The description of a peptide as non-naturally occurring is intended to exclude the naturally occurring form of the parent peptide.

[0099] Compositions comprising lacritin peptides or biologically active fragments or derivatives thereof find use in treating diseases, disorders, and conditions related to the ocular surface, including dry eye. Thus, in one embodiment, compositions comprising lacritin polypeptides are used to treat such diseases, disorders, and conditions.

[0100] According to one embodiment, a composition is provided comprising a heparinase peptide, a non-natural peptide, or a peptidomimetic derivative thereof, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NO: 4, or a sequence that differs from SEQ ID NO: 4 by 1, 2, 3, 4, or 5 amino acids, and homologs and fragments thereof. In one embodiment, a heparinase peptide is provided that differs from SEQ ID NO: 4 by 1, 2, 3, 4, or 5 amino acid modifications. In one embodiment, the amino acid modifications are amino acid substitutions, and in one embodiment, the substitutions are conservative amino acid substitutions.

[0101] In some embodiments, a peptide of the present disclosure comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 4, or a fragment or homolog thereof.

[0102] In some embodiments, a peptide of the present disclosure comprises a non-naturally occurring amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 4 or a peptidomimetic derivative of SEQ ID NO: 4. The description of a peptide as non-naturally occurring is intended to exclude the naturally occurring form of the parent peptide.

[0103] In one embodiment, the derivatives described herein comprise an amino acid sequence in which one, two, or three amino acids have been deleted, substituted, or added compared to the parent peptide, so long as the modified peptide has activity equivalent to that of the peptide having the amino acid sequence described above.

[0104] In another embodiment, novel isolated polypeptides are provided having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or a biologically active fragment, homolog, or derivative thereof. In one embodiment, the polypeptide has the amino acid sequence of SEQ ID NO:5, or a biologically active fragment, homolog, or derivative thereof. In another embodiment, the polypeptide has the amino acid sequence of SEQ ID NO:6. In another embodiment, the polypeptide has the amino acid sequence of SEQ ID NO:7, or a biologically active fragment, homolog, or derivative thereof. In another embodiment, the polypeptide has the amino acid sequence of SEQ ID NO:8, or a biologically active fragment, homolog, or derivative thereof.

[0105] In another embodiment, the present invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or a biologically active fragment, homolog, or derivative thereof, for use in therapy. In one embodiment, the present invention provides a purified ... consisting of the amino acid sequence of SEQ ID NO:7, for use in treating a disease, injury, or condition involving the ocular surface.

[0106] In another embodiment, the present invention provides use of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or a biologically active fragment, homolog, or derivative thereof, for the manufacture of a medicament for the treatment of a disease, injury, or condition related to the ocular surface, or any of the indications listed herein. In one embodiment, the polypeptide is a purified polypeptide consisting of the amino acid sequence of SEQ ID NO:7.

[0107] In another embodiment, the present invention provides a novel pharmaceutical composition comprising a therapeutically effective amount of at least one polypeptide comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 or a biologically active fragment, homolog or derivative thereof, wherein the composition is suitable for topical administration to the ocular surface of a subject.

[0108] In another embodiment, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO: 5, or a biologically active fragment, homolog, or derivative thereof. In another embodiment, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO: 6, or a biologically active fragment, homolog, or derivative thereof. In another embodiment, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO: 7, or a biologically active fragment, homolog, or derivative thereof. In another embodiment, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO: 8, or a biologically active fragment, homolog, or derivative thereof.

[0109] In one embodiment, the composition of the present invention further comprises a carrier. In one embodiment, the carrier is buffered saline. In one embodiment, the composition of the present invention is a pharmaceutical composition. In one embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. In one embodiment, the carrier is buffered saline. In one embodiment, the pharmaceutical composition of the present invention further comprises at least one additional therapeutic agent. In another embodiment, the composition further comprises buffered saline. In another embodiment, the buffer is phosphate buffer. In another embodiment, the buffer is selected from sodium phosphate, disodium phosphate, potassium phosphate, dipotassium phosphate, and combinations thereof.

[0110] In another embodiment, the composition further comprises a salt selected from NaCl and KCl. In another embodiment, the pH of the solution is selected from 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, and 8. In another embodiment, the pH is 7.4.

[0111] An example of a buffered saline solution for the present invention comprises HO and the following components: [Table 2]

[0112] In another embodiment, the present invention provides a novel method for treating dry eye, comprising contacting the ocular surface of a subject in need thereof with a composition of the present invention. According to one embodiment, treatment with lacritin based on the compositions described herein produces one or more of the following effects: a) The treatment restores or increases tearing; b) the treatment restores or increases tearing without causing or increasing inflammation; c) The treatment restores basal tearing; d) The treatment suppresses lacrimal gland inflammation; e) The treatment reduces the eye's sensitivity to corneal staining; f) The treatment decreases tear osmolality; g) The treatment improves ocular surface health; h) The procedure stimulates the lacrimal glands; i) The treatment stimulates the meibomian glands; j) The treatment stimulates conjunctival goblet cells; k) The procedure stimulates the corneal sensory nerves; l) the treatment increases the level of the shed, deglycanized 90 kDa form of syndecan-1 in the tears of treated subjects; m) the treatment reduces the level of the 25 kDa fragment of the SDC-1 ectodomain in the tears of the treated subject; n) the treatment reduces the level of an inactive lacritin-C splice variant in the tears of the treated subject; o) the treatment increases the level of latent heparanase in the tears of the treated subject; and p) The treatment reduces the level of activated heparanase in the tears of the treated subject. Patients suitable for treatment with the lacritin containing composition include those with one or more of the following conditions: a) the subject's pre-treatment tears contain reduced levels of 90 kDa deglycanated SDC-1 compared to normal, non-dry eye tears; b) the subject's pre-treatment tears contain elevated levels of 25 kDa SDC-1 compared to normal, non-dry eye tears; c) the subject's pre-treatment tears contain elevated levels of the inactive lacritin-C splice variant compared to normal, non-dry eye tears; d) the subject's pre-treatment tear fluid contains reduced levels of latent heparanase compared to normal, non-dry eye tear fluid; e) the subject's pre-treatment tear fluid contains elevated levels of activated heparanase compared to normal, non-dry eye tear fluid; and f) A subject is recovering from PRK (photorefractive keratectomy) or LASIK (laser in situ keratomileusis) surgery or other surgical procedure on the eye, regardless of the time prior to the surgery, including any subject who has had PRK or LASIK surgery and is suffering from dry eye. In one embodiment, subjects who have had PRK or LASIK surgery within the past day, month, six months, year, or years may benefit from treatment with lacritin, including the formulations described herein.

[0113] According to one embodiment, a method for identifying a subject suffering from insufficient tear fluid to moisturize and nourish the eye, not producing enough tears, or having poor tear quality. Latent heparanase / active heparanase; 90kDa deglycanated SDC-1; 25kDa SDC-1; and Inactive lacritin-C splice variant wherein the concentrations of latent heparanase or active heparanase and 90 kDa deglycanated SDC-1 are measured, and a decrease in the level of latent heparanase or an increase in active heparanase (compared to the level present in tears from a normal eye) and / or a decrease in the level of 90 kDa deglycanated SDC-1 (compared to the level present in tears from a normal eye) and / or detection of 25 kDa SDC-1, and / or detection of an inactive lacritin-C splice variant in the tear sample identifies the subject as having dry eye. Detection of any one of the four conditions, or any combination thereof, in an individual is indicative of a subject suffering from dry eye who would benefit from topical administration of a composition comprising a lacritin polypeptide, for example, including the lacritin fragment of SEQ ID NO: 7.

[0114] According to one embodiment, a tear fluid sample is obtained from a subject and the concentrations of 90 kDa deglycanated SDC-1 and 25 kDa SDC-1 are measured. A decrease in the level of 90 kDa deglycanated SDC-1, together with an increase in the level of 25 kDa SDC-1, compared to the concentrations of these peptides from normal eyes, identifies the subject as having dry eye. In one embodiment, a tear fluid sample is obtained from a subject and the sample is tested for the presence of 25 kDa SDC-1, wherein detection of 25 kDa SDC-1 identifies the subject as having dry eye. In one embodiment, a tear fluid sample is obtained from a subject and the sample is tested for the presence of an inactive lacritin-C splice variant, wherein detection of an inactive lacritin-C splice variant identifies the subject as having dry eye. In one embodiment, a tear fluid sample is obtained from a subject and the sample is tested for the presence of 25 kDa SDC-1 and the inactive lacritin-C splice variant, wherein detection of 25 kDa SDC-1 and the inactive lacritin-C splice variant identifies the subject as having dry eye.

[0115] Advantageously, these markers of dry eye can serve as a basis for identifying subjects who will benefit from lacritin treatment.Therefore, in one embodiment, a method for treating dry eye is provided, wherein the first step comprises identifying these subjects who are suitable for treatment.In one embodiment, a method for treating a subject for dry eye comprises obtaining a tear sample from the subject and detecting the presence of at least one protein selected from the group consisting of latent heparanase / active heparanase, 90kDa deglycanated SDC-1, 25kDa SDC-1 and inactive lacritin-C splice variant, wherein a decrease in latent heparanase level or an increase in active heparanase (compared to the level present in tears from normal eyes), a decrease in 90kDa deglycanated SDC-1 level (compared to tears from normal eyes), detection of 25kDa SDC-1, and / or detection of inactive lacritin-C splice variant identifies a subject with dry eye. A subject identified as having dry eye based on detected levels of latent heparanase, active heparanase, 90 kDa deglycanated SDC-1, 25 kDa SDC-1, and / or the inactive lacritin-C splice variant is then administered a composition comprising a lacritin polypeptide, for example, including the peptide of SEQ ID NO: 7. More specifically, the subject's ocular surface is contacted with a pharmaceutical composition comprising lacritin or a biologically active fragment thereof.

[0116] According to one embodiment, a subject identified as suffering from dry eye is KQFIENGSEFAQKLLKKFS(SEQ ID NO:5); KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7); KQFIENGSEFANKLLKKFS (SEQ ID NO: 6); and KQFIENGSEFANKLLKKFSLLKPWA (SEQ ID NO: 8), or a derivative thereof that differs from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 by one or two amino acid substitutions. In one embodiment, the biologically active fragment of lacritin consists of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7).

[0117] According to one embodiment, subjects suffering from dry eye that are treatable with lacritin therapy are identified by testing the subject's tears for the presence of lacritin monomer, where a selective reduction in the monomer compared to a control is indicative of dry eye. The control is a subject (or group of subjects) that does not suffer from dry eye. In another embodiment, subjects suffering from dry eye that are treatable with lacritin therapy are identified by testing the subject's tears for the presence of 90 kDa deglycanated SDC-1, where detection of normal levels of 90 kDa deglycanated SDC-1 is not indicative of dry eye. In another embodiment, subjects suffering from dry eye that are treatable with lacritin therapy are identified by collecting the subject's tears and separating tear proteins by weight. In one embodiment, tear proteins are separated using SDS-PAGE.

[0118] In another embodiment, the test is performed by contacting the subject's tears with a test strip containing a substance capable of detecting the presence of 25 kDa SDC-1. In one embodiment, the substance is an antibody, suitably a monoclonal antibody.

[0119] In another embodiment, the test is performed by contacting the subject's tears with a test strip containing a substance capable of detecting the presence of 90 kDa deglycanated SDC-1. In one embodiment, the substance is an antibody, suitably a monoclonal antibody.

[0120] In another embodiment, tears are tested for the presence of 25 kDa SDC-1 and 90 kDa deglycanated SDC-1, wherein detection of normal levels of 90 kDa deglycanated SDC-1 does not indicate dry eye, and the presence of 25 kDa SDC-1 indicates dry eye. In another embodiment, the method for identifying a patient suffering from dry eye further comprises treating the subject with a composition of the present invention. In one embodiment, the ocular surface of a subject found to have 25 kDa SDC-1 in tears is treated with a lacritin or lacritin fragment containing composition.

[0121] In another embodiment, the present invention provides a novel method for identifying a subject with dry eye, comprising testing the subject's tears for the presence of 25 kDa SDC-1, wherein the presence of 25 kDa SDC-1 is indicative of dry eye. In one embodiment, the testing is performed by collecting the subject's tears and separating the proteins of the tears based on weight. In one embodiment, separation of peptides is performed using SDS-PAGE.

[0122] In another embodiment, the method provides for testing a subject's tears for the presence of an inactive lacritin-C splice variant, wherein the presence of an inactive lacritin-C splice variant indicates dry eye. In one embodiment, the presence of an inactive lacritin-C splice variant is detected by collecting the subject's tears and separating the tear proteins by weight. In one embodiment, the tear proteins are separated using SDS-PAGE. In another embodiment, the test is performed by contacting the subject's tears with a test strip containing a substance capable of detecting the presence of an inactive lacritin-C splice variant. In one embodiment, the substance is an antibody, suitably a monoclonal antibody. In a further embodiment, a method of treatment is provided comprising contacting the ocular surface of a subject found to have an inactive lacritin-C splice variant in their tears with a composition of the present invention.

[0123] In another embodiment, a novel method for identifying a subject with dry eye comprises testing the subject's tears for the presence of active and latent heparanase, wherein the presence of increased active heparanase or decreased latent heparanase is indicative of dry eye. In one embodiment, the test is performed by collecting the subject's tears, separating the tear proteins by weight, and blotting with an anti-heparanase antibody that can detect latent and active heparanase.

[0124] In one embodiment, a subject suffering from evaporative dry eye, corneal inflammation or corneal ulceration can be treated by contacting the cells of the subject in need thereof with a lacritin polypeptide comprising a composition described herein.

[0125] In another embodiment, the present invention provides a novel method for enhancing the proliferation of human corneal epithelial cells or lacrimal acinar cells, wherein the method comprises contacting cells in a subject in need thereof with a composition of the present invention. In one embodiment, the lacritin peptide compositions described herein are used to enhance the proliferation of corneal epithelial cells in a subject, enhance the proliferation of lacrimal acinar cells in a subject, or inhibit epithelial cell apoptosis or other forms of epithelial cell death. In one embodiment, the method comprises contacting cells in a subject in need thereof with a lacritin peptide composition described herein, wherein the cells are selected from corneal cells, conjunctival cells, or a combination of both.

[0126] In another embodiment, the epithelial cells contacted with the lacritin peptide have been exposed to an injury. In one embodiment, the lacritin peptide consists of the sequence of SEQ ID NO: 7. In one embodiment, the injury is selected from the group consisting of blepharitis, dry eye, conjunctivitis, Sjogren's syndrome, corneal abrasion, ulceration, bacterial infection, direct trauma, surgery, radiant energy, ionizing energy, viral infection, fungal infection, parasitic infection, keratitis, systemic skin disorder, collagen vascular disease, Reiter's disease, and Behcet's disease.

[0127] In another embodiment, provided is a method for treating lysosomal clearing disease, wherein the method comprises contacting the ocular surface of subject with the composition of the present disclosure.In one embodiment, the disease is selected from glaucoma and age-related macular degeneration (AMD).In one embodiment, the method comprises contacting the ocular surface of subject with the composition comprising lacritin peptide, wherein the peptide consists of the sequence of SEQ ID NO:7.

[0128] In another embodiment, the present invention relates to the treatment of lysosomal clearing. Examples of such diseases include glaucoma and age-related macular degeneration (AMD). Without wishing to be bound by scientific theory, lacritin is thought to induce autophagic trapping and lysosomal degradation of intracellularly activated (toxic) proteins in stressed cells. In AMD, "drusen" formation occurs both intracellularly within retinal pigment epithelial cells and extracellularly near these cells. It is predicted that topical administration of a polypeptide of the present invention (e.g., lacritin, or a polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8) deep into the eye will stimulate RPE autophagy and dramatically reduce drusen. In open-angle glaucoma, stress in trabecular meshwork cells causes the formation of accumulating intracellular material. Chronically increased autophagy is unhealthy for cells. Instead, lacritin forces a rapid, temporary bolus of accelerated autophagy sufficient to remove the problematic accumulating proteins. Autophagy then returns to baseline. The polypeptides of the present disclosure are expected to gain access to these cells.

[0129] In another embodiment, the present invention provides a novel bactericidal composition. According to one embodiment, a bactericidal composition is provided comprising a C-terminal fragment of lacritin selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or a derivative thereof that differs from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 by one, two, or three amino acid substitutions. In one embodiment, the composition is suitable for topical administration to the ocular surface of a subject. In one embodiment, the lacritin derivative that differs from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 by one, two, or three amino acid substitutions has an amino acid substitution at a position selected from positions 4, 6, 8, 10, 17, and 19 relative to the numbering of SEQ ID NO:7. These positions show variability among the highly conserved C-terminal regions of primate species (see Figure 9). In one embodiment, the lacritin derivative differs from SEQ ID NO:7 or SEQ ID NO:8 by one or two amino acid substitutions at positions 4 and / or 19 relative to the numbering of SEQ ID NO:7. In one embodiment, the amino acid substitution is a conservative amino acid substitution. According to one embodiment, there is provided a bactericidal composition comprising a C-terminal fragment of lacritin selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. According to one embodiment, there is provided a bactericidal composition comprising a C-terminal fragment of lacritin selected from SEQ ID NO: 7 or SEQ ID NO: 8, and in one embodiment, the C-terminal fragment of lacritin consists of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7).

[0130] In one embodiment, a bactericidal composition is provided comprising a first antibacterial agent, wherein the antibacterial agent is a polypeptide comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or a biologically active fragment, homolog, or derivative thereof, and the composition is suitable for topical administration to the ocular surface of a subject. In one embodiment, the polypeptide has the amino acid sequence of SEQ ID NO:5. In another embodiment, the polypeptide has the amino acid sequence of SEQ ID NO:6. In another embodiment, the polypeptide has the amino acid sequence of SEQ ID NO:7. In another embodiment, the polypeptide has the amino acid sequence of SEQ ID NO:8. In one embodiment, the bactericidal composition further comprises a bactericidal acceptable carrier.

[0131] In one embodiment, the antiseptic composition further comprises a second antibacterial agent. In one embodiment, the composition further comprises an antibacterial agent. Suitable ophthalmic antibacterial agents are known to those skilled in the art and include those described in U.S. Patent Nos. 5,300,296, 6,316,669, 6,365,636 and 6,592,907, the disclosures of which are incorporated herein by reference. Examples of antibacterial agents suitable for use in the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, chlorobutanol, chlorhexidine diglucuronate or diacetate, methyl and propyl hydroxybenzoates (parabens), phenylethyl alcohol, phenylmercuric acetate or nitrate, sorbic acid, and thimerosal.

[0132] In one embodiment, a second antibacterial agent is present in the antibacterial composition, and in one embodiment, the second antibacterial agent is one that is naturally occurring in the mammalian eye. In one embodiment, the second antibacterial agent is lysozyme. According to one embodiment, the antibacterial composition comprises a C-terminal fragment of lacritin and a second antibacterial agent, wherein the ratio of the C-terminal fragment of lacritin to the second antibacterial agent is at least 2:1. In one embodiment, the antibacterial composition comprises a C-terminal fragment of lacritin and lysozyme, wherein the weight ratio of lysozyme to the C-terminal fragment of lacritin is 4:1 to 3:1. In one embodiment, the C-terminal fragment of lacritin consists of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7).

[0133] According to one embodiment, a method for treating an eye infection is provided. The method comprises topically administering to the eye a composition comprising a lacritin polypeptide. In another embodiment, the present invention provides a novel method for treating a corneal infection, comprising contacting the cornea of ​​a subject in need thereof with a bactericidal composition of the present invention. In one embodiment, a peptide consisting of the sequence KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7) is used in the manufacture of a medicament for treating dry eye or treating a corneal infection.

[0134] In another embodiment, a method of treating a subject suffering from dry eye by contacting the ocular surface of the subject with a composition of the present invention and active heparanase in tears has been found.

[0135] In another embodiment, the present invention provides a novel container comprising a composition of the present invention, wherein the composition is in the form of eye drops and is in an amount sufficient for one dose. In another embodiment, the composition is in the form of eye drops and is in an amount sufficient for one to two doses. In another embodiment, the composition is in the form of eye drops and is in an amount sufficient for up to one week, two weeks, three weeks, or four weeks. In another embodiment, the container is in the form of a single-use ampoule, a bottle configured for administering eye drops of the composition, or a bottle comprising a body and a cap, wherein an eye dropper connects to the cap or part of the cap.

[0136] U.S. Patent Nos. 7,648,964, 7,459,440, 7,320,870 and 7,932,227, and WO 98 / 27205 (Jacobs et al., published June 25, 1998), Sanghi et al., 2001, J. Mol. Biol., 310:127, Wang et al. al., 2006, J. Cell Biol., 174(5):689-700, Epub 2006 Aug 21, Ma et al., J. Cell Biol., 2006, 174:7:1097-1106, Zhang et al., J. Biol. Chem., 2013, 288(17):12090-101: Epub 2013 Mar The invention can also be practiced using the methods described in 15, the contents of which are incorporated herein by reference in their entirety.

[0137] Various aspects and embodiments of the invention are described in further detail below.

[0138] According to one embodiment, a novel mechanism for the molecular identification of dry eye disease is coupled with restorative therapies that address the cause. In one embodiment, a method for identifying dry eye involves the discovery that an approximately 90 kDa deglycanized syndecan-1 fragment is abundant in the tears of normal individuals but not in those suffering from dry eye, while an approximately 25 kDa syndecan-1 fragment is detectable in dry but not normal tears. Also disclosed herein is the discovery that topical lacritin, an agonist of deglycanized syndecan-1, sensitizes corneal sensory nerves to ocular surface dryness and increases the moistening response of the nerve. Accordingly, one embodiment of the present invention relates to a method for identifying dry eye by detecting abnormally reduced levels of approximately 90 kDa and / or the presence of 25 kDa syndecan-1 in tears. Another embodiment relates to a method for increasing the dryness and wetting response of corneal nerves by administering topical lacritin or a lacritin fragment, synthetic peptide, or mimetic.

[0139] Current tear replacement products are not widely used by patients, in part because the relief they provide is extremely short-lived (less than 15 minutes). Examples of tear replacement approaches include buffered isotonic saline solutions and aqueous solutions containing water-soluble polymers that make the solution more viscous and less likely to flow from the eye. Tear reconstitution has also been attempted by providing one or more components of the tear film, such as phospholipids and oils. Examples of these treatment approaches are described in U.S. Pat. No. 4,131,651 (Shah et al.), U.S. Pat. No. 4,370,325 (Packman), U.S. Pat. No. 4,409,205 (Shively), U.S. Pat. Nos. 4,744,980 and 4,883,658 (Holly), U.S. Pat. No. 4,914,088 (Glonek), U.S. Pat. No. 5,075,104 (Gressel et al.), and U.S. Pat. No. 5,294,607 (Glonek et al.), the disclosures of which are incorporated herein by reference. Existing ophthalmic formulations may also contain TGF-β, corticosteroids, or androgens, all of which are non-specific to the eye and have systemic effects. In contrast, lacritin is highly ocular-specific and is a natural component of human tears and tear film.

[0140] Ophthalmic formulations containing lacritin or its fragments, analogs, or derivatives (e.g., artificial tears containing lacritin) are highly desirable due to the activity of lacritin and its localized effects. According to one embodiment of the present invention, compositions containing lacritin or its bioactive fragments are used to enhance corneal wound healing and / or treat patients with insufficient tear flow. The lacritin compositions of the present invention can be formulated with standard ophthalmic ingredients, preferably as solutions, suspensions, and other dosage forms for topical administration. Aqueous solutions are generally preferred based on ease of formulation, biocompatibility (particularly considering the condition to be treated, e.g., dry eye-type diseases and disorders), and the patient's ability to easily administer such compositions by instilling one to two drops of the solution into the affected eye. However, the compositions can also be suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions.

[0141] The compositions of the present invention may contain surfactants, preservatives, antioxidants, tonicity agents, buffers, preservatives, cosolvents, and viscosity-building agents. Various surfactants useful in topical ophthalmic formulations may be used in the compositions. These surfactants may help prevent chemical degradation of lacritin and may also prevent lacritin from binding to the container in which the composition is packaged. Examples of surfactants include, but are not limited to, Cremophor. TM EL, polyoxyl 20 cetostearyl ether, polyoxyl 40 hydrogenated castor oil, polyoxyl 23 lauryl ether, and poloxamer 407 may be used in the compositions. Antioxidants may be added to the compositions of the present invention to protect the lacritin polypeptide from oxidation during storage. Examples of antioxidants include, but are not limited to, vitamin E and its analogs, ascorbic acid and derivatives, and butylhydroxyanisole (BHA).

[0142] Existing artificial tear formulations can also be used as pharmaceutically acceptable carriers for lacritin active agents. Thus, in one embodiment, lacritin polypeptides are used to improve existing artificial tear products for dry eye syndrome and to develop products that aid in corneal wound healing. Examples of artificial tear compositions useful as carriers include Tears Naturale TM , Tears Naturale II TM , Tears Naturale Free TM , and Bion Tears TMCommercially available phospholipid carrier formulations include, but are not limited to, those described in U.S. Patent No. 4,804,539 (Guo et al.), U.S. Patent No. 4,883,658 (Holly), U.S. Patent No. 4,914,088 (Glonek), U.S. Patent No. 5,075,104 (Gressel et al.), U.S. Patent No. 5,278,151 (Korb et al.), U.S. Patent No. 5,294,607 (Glonek et al.), U.S. Patent No. 5,371,108 (Korb et al.), and U.S. Patent No. 5,578,586 (Glonek et al.); the foregoing patents are incorporated herein by reference insofar as they disclose phospholipid compositions useful as phospholipid carriers of the present invention. According to one embodiment, a topical ophthalmic formulation is provided comprising a lacritin peptide consisting of the sequence of SEQ ID NO:7 and a pharmaceutically acceptable carrier. In one embodiment, the composition further comprises a phospholipid. In an alternative embodiment, the composition further comprises a surfactant, a preservative, an antioxidant, a tonicity agent, a buffer, a preservative, a co-solvent and / or a viscosity increasing agent.

[0143] Other compounds may also be added to the ophthalmic compositions of the present disclosure to increase the viscosity of the carrier. Examples of viscosity-increasing agents include, but are not limited to, polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextran, various polymers of the cellulose family; vinyl polymers; and acrylic acid polymers. Generally, phospholipid carrier or artificial tear carrier compositions exhibit a viscosity of 1 to 400 centipoise ("cps"). Preferred compositions containing artificial tears or phospholipid carriers exhibit a viscosity of about 25 cps.

[0144] Topical ophthalmic products are typically packaged in multi-dose form. Therefore, preservatives are necessary to prevent microbial contamination during use. Suitable preservatives include benzalkonium chloride, chlorobutanol, benzododecinium bromide, methylparaben, propylparaben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1, or other substances known to those skilled in the art. Such preservatives are typically used at levels of 0.001-1.0% w / v. The unit dose compositions of the present invention are sterile and typically unpreserved. Therefore, such compositions generally do not contain preservatives.

[0145] Because the gene promoter that regulates lacritin gene expression is most specific to any of the lacrimal gland genes described above, the regulatory elements of this gene can be used to express other gene products in the eye. In particular, the lacritin gene promoter can be operably linked to a wide range of exogenous genes to regulate the expression of gene products in the lacrimal gland and / or used as gene therapy to treat dry eye syndrome.

[0146] The peptides of the present disclosure can be readily prepared by standard, well-established techniques, such as solid-phase peptide synthesis (SPPS), as described in "Solid Phase Peptide Synthesis," 2nd Edition, by Stewart et al., 1984, Pierce Chemical Company, Rockford, Illinois; and "The Practice of Peptide Synthesis," by Bodanszky and Bodanszky, 1984, Springer-Verlag, New York. First, a suitably protected amino acid residue is attached via its carboxyl group to a derivatized, insoluble polymer support, such as cross-linked polystyrene or polyamide resin. "Suitably protected" refers to the presence of protecting groups on both the α-amino group of the amino acid and any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents, and reaction conditions used throughout the synthesis and can be removed under conditions that do not affect the final peptide product. Stepwise synthesis of oligopeptides is carried out by removing the N-protecting group from the first amino acid and attaching it to the carboxyl terminus of the next amino acid in the desired peptide sequence, which is also suitably protected. The carboxyl of the incoming amino acid can then be activated and reacted with the N-terminus of the support-bound amino acid by formation into a reactive group, such as a carbodiimide, symmetrical anhydride, or "active ester" group such as a hydroxybenzotriazole or pentafluorophenyl ester.

[0147] Examples of solid phase peptide synthesis methods include the BOC method, which utilizes tert-butyloxycarbonyl as the α-amino protecting group, and the FMOC method, which utilizes 9-fluorenylmethyloxycarbonyl to protect the α-amino of an amino acid residue, both of which are well known to those skilled in the art.

[0148] Incorporation of N- and / or C-blocking groups can also be achieved using protocols conventional for solid-phase peptide synthesis. For incorporation of a C-terminal blocking group, for example, synthesis of the desired peptide is typically accomplished using a support resin as the solid phase that has been chemically modified so that cleavage from the resin yields a peptide with the desired C-terminal blocking group. To provide a peptide with a primary amino blocking group at the C-terminus, synthesis is performed, for example, using p-methylbenzhydrylamine (MBHA) resin, such that treatment with hydrofluoric acid liberates the desired C-terminally amidated peptide upon completion of peptide synthesis. Similarly, incorporation of an N-methylamine blocking group at the C-terminus is achieved using an N-methylaminoethyl-derivatized DVB resin, which, upon HF treatment, liberates a peptide with an N-methylamidated C-terminus. Blocking of the C-terminus by esterification can also be achieved using conventional procedures. This requires the use of a resin / blocking group combination that allows for the release of the side-chain peptide from the resin to allow subsequent reaction with the desired alcohol to form an ester functionality. An FMOC protecting group in combination with a DVB resin derivatized with a methoxyalkoxybenzyl or equivalent linker can be used for this purpose, with cleavage from the support occurring with TFA in dichloromethane. Esterification of an appropriately activated carboxyl function, for example with DCC, can then proceed by addition of the desired alcohol, followed by deprotection and isolation of the esterified peptide product.

[0149] Incorporation of an N-terminal blocking group can be achieved while the synthesized peptide is still attached to the resin, for example, by treatment with an appropriate anhydride and nitrile. To incorporate an acetyl-blocking group at the N-terminus, for example, the resin-bound peptide can be treated with 20% acetic anhydride in acetonitrile. The N-blocked peptide product can then be cleaved from the resin, deprotected, and subsequently isolated.

[0150] To confirm that a peptide obtained from either chemical or biological synthesis techniques is the desired peptide, analysis of the peptide composition should be performed. The amino acid composition analysis can be performed using high-resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively or additionally, the amino acid content of a peptide can be confirmed by hydrolyzing the peptide in an acidic aqueous solution and separating, identifying, and quantifying the components of the mixture using HPLC or an amino acid analyzer. To unambiguously determine the sequence of a peptide, a protein sequenator can be used to decompose the peptide in sequence and identify the amino acids in order.

[0151] Prior to its use, the peptide is purified to remove contaminants. In this regard, it will be understood that the peptide is purified to meet the standards set by the appropriate regulatory authorities. To achieve the required level of purity, any one of a number of conventional purification procedures can be used, including, for example, reverse-phase high-performance liquid chromatography (HPLC) using an alkylated silica column, such as C4-, C8-, or C18-silica. A gradient mobile phase of increasing organic content can be used to achieve purification, for example, typically using acetonitrile in an aqueous buffer containing a small amount of trifluoroacetic acid. Ion exchange chromatography can also be used to separate peptides based on their charge.

[0152] It will of course be understood that the peptides, or antibodies, derivatives, or fragments thereof, may incorporate amino acid residues that are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e., chemical substituents, suitable to protect and / or stabilize the N- and C-termini from "undesired degradation" (a term meant to encompass any type of enzymatic, chemical, or biochemical degradation of the compound at that terminus that may affect the function of the compound, i.e., sequence degradation of the compound at that terminus).

[0153] Blocking groups include protecting groups conventionally used in the field of peptide chemistry that do not adversely affect the activity of peptides in vivo. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, and substituted forms thereof, such as the acetamidomethyl (Acm) group. Deamino analogs of amino acids are also useful N-terminal blocking groups, which can be either attached to the N-terminus of a peptide or used in place of the N-terminal residue. Suitable C-terminal blocking groups, in which the C-terminal carboxyl group is either incorporated or not, include esters, ketones, or amides. Examples of C-terminal blocking groups include alkyl groups that form esters or ketones, particularly lower alkyl groups such as methyl, ethyl, and propyl, and amino groups that form amides, such as primary amines (-NH2), as well as mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, and methylethylamino. Decarboxylated amino acid analogs, such as agmatine, are also useful C-terminal blocking groups and can be either attached to or used in place of the C-terminal residue of a peptide. Furthermore, it will be appreciated that the free amino and carboxyl groups at the termini can be removed entirely from the peptide to yield its deaminated and decarboxylated form without any effect on peptide activity.

[0154] Other modifications can also be incorporated without adversely affecting activity, including, but not limited to, the substitution of one or more amino acids in their natural L-isomer form with amino acids in their D-isomer form. Thus, the peptides can contain one or more D-amino acid residues, or can contain amino acids that are all D-form. Retro-reverse peptides in accordance with the invention, e.g., in which all amino acids are substituted with D-amino acid forms, are also contemplated.

[0155] Acid addition salts of the present invention are also contemplated as functional equivalents. Thus, peptides according to the present invention that have been treated with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or an organic acid, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc., to provide a water-soluble salt of the peptide are suitable for use in the present invention.

[0156] They may differ from naturally occurring proteins or peptides by differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes can be made that alter the primary sequence of the protein or peptide, but do not normally alter its function. Thus, 10 or more conservative amino acid changes typically do not affect peptide function. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; Lysine, arginine; Phenylalanine, tyrosine

[0157] Modifications (which do not normally alter the primary sequence) include in vivo or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are glycosylation modifications, e.g., made by modifying the glycosylation pattern of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation, e.g., mammalian glycosylation or deglycosylation enzymes. Also encompassed are sequences having phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0158] Also included are polypeptides or antibody fragments that have been modified using conventional molecular biology techniques to improve their resistance to proteolysis, maximize their solubility, or make them more suitable as therapeutic substances.Such polypeptide analogs include those that contain residues other than naturally occurring L-amino acids, such as D-amino acids or unnatural synthetic amino acids.The peptides of the present invention are not limited to the products of any of the specific exemplary processes listed herein.

[0159] Those skilled in the art will recognize that, in general, amino acid substitutions in peptides typically involve replacing an amino acid with another amino acid that has relatively similar properties (i.e., conservative amino acid substitutions). The properties of various amino acids and the effects of amino acid substitutions on protein structure and function have been the subject of extensive study and knowledge in the art.

[0160] For example, the following isosteric and / or conservative amino acid changes can be made to a parent polypeptide sequence with the expectation that the resulting polypeptide will have a similar or improved profile of the properties described above.

[0161] Alkyl-substituted hydrophobic amino acid substitutions: include alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid, S-cyclohexylalanine, or other similar alpha amino acids substituted with aliphatic side chains of C1-10 carbons, including branched, cyclic, and straight-chain alkyl, alkenyl, or alkynyl substitutions.

[0162] Aromatic substituted hydrophobic amino acid substitutions include: phenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy substituted versions of the aromatic amino acids listed above, examples of which are 2-, 3-, or 4-aminophenylalanine, 2-, 3-, or 4-chlorophenylalanine. 2'-, 3'-, or 4'-amino-, 2'-, 3'-, or 4'-chloro-, 2-, 3-, or 4-biphenylalanine; 2'-, 3'-, or 4'-methyl-, 2-, 3-, or 4-biphenylalanine; 2'-, 3'-, or 4'-methyl-, 2-, 3-, or 4-biphenylalanine; 4'-methyl-, 2-, 3-, or 4-biphenylalanine; and 2- or 3-pyridylalanine.

[0163] Substitutions for amino acids containing basic functional groups include: arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, and alkyl-, alkenyl-, or aryl-substituted (C1-C10 branched, linear, or cyclic) derivatives of the foregoing amino acids, whether the substituent is on a heteroatom (e.g., the α-nitrogen or one or more distal nitrogen atoms) or on the α-carbon, e.g., in the pro-R position. Illustrative compounds include N-ε-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, and N,N-γ,γ'-diethyl-homoarginine. Also included are compounds in which an alkyl group occupies the pro-R position of the α-carbon, such as α-methylarginine, α-methyl-2,3-diaminopropionic acid, α-methylhistidine, and α-methylornithine. Also included are amides formed from alkyl, aromatic, heteroaromatic (where a heteroaromatic group has one or more nitrogen, oxygen, or sulfur atoms, alone or in combination) carboxylic acids or any of the many well-known activated derivatives (e.g., acid chlorides, active esters, activated azolides, and related derivatives) with lysine, ornithine, or 2,3-diaminopropionic acid.

[0164] Acidic amino acid substitutions: include aspartic acid, glutamic acid, homoglutamic acid, tyrosine, 2,4-diaminopriopionic acid, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of ornithine or lysine, and tetrazole-substituted alkyl amino acids.

[0165] Substitutions of side chain amide residues: include asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine.

[0166] Substitutions for hydroxyl-containing amino acids include: serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl- or aromatic-substituted derivatives of serine or threonine. It is also understood that an amino acid within each category listed above can be substituted for another amino acid in the same group.

[0167] For example, one can consider the hydropathic index of amino acids (Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relative hydropathic character of amino acids contributes to the secondary structure of the resulting protein, which in turn determines the interactions of that protein with other molecules. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), which are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). When making conservative substitutions, it is preferable to use amino acids whose hydropathic index is within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0168] Amino acid substitutions may take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554,101). Amino acid residues have been assigned hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0); glutamic acid (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 +-0.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of an amino acid with another amino acid having a similar hydrophilicity is preferred.

[0169] Other considerations include the size of the amino acid side chain. For example, it would generally be undesirable to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, such as tryptophan or tyrosine. The effect of various amino acid residues on protein secondary structure is also a consideration. Experimental studies have determined the effects of different amino acid residues on the propensity of protein domains to adopt α-helix, β-sheet, or reverse turn secondary structures and are known in the art (see, e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys. J., 26:367-384).

[0170] Based on such considerations and extensive experimental studies, conservative amino acid substitution tables have been compiled and are known in the art. For example, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Or: Ala (A) leu, ile, val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu(L)val,met,ala,phe,ile;Lys(K)gln,asn,arg;Met(M)phe,ile,leu;Phe(F)leu,val,ile,ala,tyr;Pr o(P)ala;Ser(S), thr;Thr(T)ser;Trp(W)phe, tyr;Tyr(Y)trp, phe, thr, ser;Val(V)ile, leu, met, phe, ala.

[0171] Other considerations for amino acid substitutions include whether the residue is located in the interior of the protein or whether it is solvent-exposed. For internal residues, conservative substitutions would include the following: Asp for Asn; Ser for Thr; Ser for Ala; Thr for Ala; Ala for Gly; Ile for Val; Val for Leu; Leu for Ile; Leu for Met; Phe for Tyr; Tyr for Trp (see, e.g., the PROWL Rockefeller University website). For solvent-exposed residues, conservative substitutions would include the following: Asp for Asn; Asp for Glu; Glu for Gln; Glu for Ala; Gly for Asn; Ala for Pro; Ala for Gly; Ala for Ser; Ala for Lys; Ser for Thr; Lys for Arg; Val for Leu; Leu for Ile; Ile for Val; Phe for Tyr. Various matrices have been constructed to aid in the selection of amino acid substitutions, including the PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix, and Risler matrix (Id.).

[0172] In determining amino acid substitutions, the presence of intermolecular or intramolecular bonds can also be taken into account, such as ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bond formation between adjacent cysteine ​​residues.

[0173] Methods for substituting any amino acid for any other amino acid in an encoded peptide sequence are well known and are no more than routine experimentation for those skilled in the art, for example by the technique of site-directed mutagenesis or by synthesizing and assembling oligonucleotides encoding the amino acid substitutions and ligating them into an expression vector construct.

[0174] The protein can be purified by following known procedures, where immunological, enzymatic or other assays are used to monitor the purification at each step of the procedure. Protein purification methods are well known in the art and are described, for example, in Deutscher et al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego.

[0175] The invention also includes a kit comprising a composition of the invention and instructions for administering the composition to a subject, hi another embodiment, the kit includes a (preferably sterile) solvent suitable for dissolving or suspending the composition of the invention prior to administration of the composition.

[0176] As used herein, the term "physiologically acceptable" ester or salt refers to an ester or salt form of an active ingredient that is compatible with any other ingredients of a pharmaceutical composition that is not harmful to the subject to which the composition is administered.

[0177] The formulations of the pharmaceutical compositions described herein can be prepared by any known method or subsequently developed in the field of pharmacology. Generally, such preparation methods include bringing the active ingredient into association with the carrier or one or more other accessory ingredients, and then, if necessary or desired, forming or packaging the product into a desired single or multiple dosage unit.

[0178] Although the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions suitable for human administration to make them suitable for administration to various animals are well understood, and a skilled pharmaceutical scientist could design and implement such modifications with no more than routine experimentation (if any). Subjects to which administration of the pharmaceutical compositions of the present invention is contemplated include, but are not limited to, humans and other primates, commercially relevant mammals such as cows, pigs, horses, sheep, cats, and dogs, and birds, including commercially relevant birds such as chickens, ducks, geese, and turkeys.

[0179] Pharmaceutical compositions useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, intravenous, topical, pulmonary, nasal, buccal, ocular, intrathecal, or another route of administration. Other contemplated formulations include engineered nanoparticles, liposomal preparations, releasable erythrocytes containing the active ingredient, and immunologically-based formulations.

[0180] The pharmaceutical composition of the present invention can be prepared, packaged or sold in bulk, as a single unit dose or as a plurality of single unit doses.As used herein, " unit dose " is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dose of active ingredient that is administered to subject, or a convenient fraction of this dose, for example, half or one-third of this dose.

[0181] The relative amounts of active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. For example, the composition may contain from 0.1% to 100% (w / w) active ingredient.

[0182] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharmaceutically active substances. Particularly contemplated additional substances include antiemetic agents and scavengers, such as cyanide and cyanate scavengers.

[0183] Controlled- or sustained-release formulations of the pharmaceutical compositions of the invention may be made using conventional techniques.

[0184] Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations, such as liniments, lotions, oil-in-water or water-in-oil emulsions, such as creams, ointments, or pastes, and solutions or suspensions. While the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent, topically administrable formulations can contain, for example, about 1% to about 10% (w / w) active ingredient. Formulations for topical administration can further include one or more additional ingredients described herein.

[0185] Pharmaceutical compositions of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such a formulation may be in the form of, for example, eye drops comprising, for example, a 0.1 to 1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such eye drops may further comprise a buffer, salt, or one or more other of the additional ingredients described herein. Other ophthalmically administrable formulations that are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

[0186] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: additives; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; biodegradable compositions, such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; excipients; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

[0187] Typically, the dose of a compound of the invention administered to a subject, preferably a human, ranges from 1 μg to about 100 g of body weight of the subject. The exact dose administered will vary depending on any number of factors, including, but not limited to, the type of subject and the type of disease state being treated, the age of the subject, and the route of administration.

[0188] The present invention further provides for the identification of subjects with dry eye. For example, proteins or peptides found in tears can be detected using a variety of methods, including, but not limited to, ELISA, immunoassays, immunofluorescence, immunohistochemistry, immunoprecipitation, and Western blot.

[0189] A kit is provided that includes a composition of the present invention and instructions for administering the composition to cells or tissues of a subject. In another embodiment, the kit includes a solvent (preferably sterile) suitable for dissolving or suspending the composition of the present invention prior to administering the compound to the subject. As used herein, "instructions" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the peptides of the present invention in the kit for alleviating the various diseases or disorders listed herein. In one embodiment, the kit provides a standard curve providing information on the concentrations of various peptides in normal, healthy eyes. Optionally, or alternatively, the instructions may describe one or more methods for alleviating a disease or disorder in cells or tissues of a subject. The instructions for the kit of the present invention may be, for example, attached to a container containing the peptide of the present invention or shipped together with the container containing the peptide. Alternatively, the instructions may be shipped separately from the container, with the instructions and the compound intended for cooperative use by the recipient. Further embodiments of the present invention are listed below: [Section 1] 1. A method for identifying a subject having dry eye, comprising: latent heparanase; 90kDa deglycanated SDC-1; 25kDa SDC-1; and inactive lacritin-C splice variant; detecting the presence of at least one protein selected from the group consisting of: a decrease in the level of latent heparanase or an increase in active heparanase compared to the level present in tears from normal eyes; Decreased levels of 90 kDa deglycanated SDC-1 compared with levels present in tears from normal eyes; Detection of 25 kDa SDC-1; and / or Detection of inactive lacritin-C splice variants However, a method for identifying a subject having dry eye. [Section 2] The method of claim 1, wherein the concentrations of 90 kDa deglycanated SDC-1 and 25 kDa SDC-1 are measured in a tear sample obtained from the subject, and a decrease in the level of 90 kDa deglycanated SDC-1 combined with an increase in the level of 25 kDa SDC-1 compared to the level present in tears from a normal eye identifies the subject as having dry eye. [Section 3] Item 10. The method of claim 1, wherein a tear sample obtained from the subject is tested for the presence of 25 kDa SDC-1, and detection of 25 kDa SDC-1 identifies the subject as having dry eye. [Section 4] Item 1. The method of item 1, wherein a tear sample obtained from the subject is tested for the presence of an inactive lacritin-C splice variant, and detection of an inactive lacritin-C splice variant identifies the subject as having dry eye. [Section 5] The method described in paragraph 1 above, wherein a tear sample obtained from the subject is tested for the presence of 25kDa SDC-1 and the inactive lacritin-C splice variant, and detection of 25kDa SDC-1 and the inactive lacritin-C splice variant identifies the subject as having dry eye. [Section 6] 1. A method of treating a subject for dry eye, comprising: a) in a tear fluid sample obtained from the subject, latent heparanase; 90kDa deglycanated SDC-1; 25kDa SDC-1; and inactive lacritin-C splice variant; identifying a patient suffering from dry eye by detecting the presence of at least one protein selected from the group consisting of: Decreased levels of latent heparanase compared with levels present in tears from normal eyes; Decreased levels of 90 kDa deglycanated SDC-1 compared with levels present in tears from normal eyes; Detection of 25 kDa SDC-1; and / or Detection of inactive lacritin-C splice variants identifying a subject with dry eye; and b) contacting the ocular surface of the subject identified in step a) with a composition comprising a lacritin polypeptide. A method comprising: [Section 7] the lacritin polypeptide is the polypeptide of SEQ ID NO: 1, or KQFIENGSEFAQKLLKKFS(SEQ ID NO:5); KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7); KQFIENGSEFANKLLKKFS (SEQ ID NO: 6); and KQFIENGSEFANKLLKKFSLLKPWA (SEQ ID NO: 8), 7. The method according to claim 6, wherein the lacritin is a biologically active fragment selected from the group consisting of: or a derivative of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 which differs from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 by one or two amino acid substitutions. [Section 8] 7. The method of claim 6, wherein the lacritin polypeptide consists of the sequence KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7). [Section 9] A pharmaceutical composition comprising a therapeutically effective amount of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7), or a peptide having a sequence that differs from KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7) by one amino acid substitution; and a pharmaceutically acceptable carrier, the composition being formulated for topical administration to the ocular surface of a subject. [Section 10] Item 10. The pharmaceutical composition according to item 9, wherein the peptide consists of the sequence KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7). [Section 11] 11. The pharmaceutical composition according to item 9 or 10, further comprising a phospholipid, a surfactant, a preservative, an antioxidant, an isotonicity agent, a buffer, a preservative, a cosolvent or a viscosity enhancer. [Section 12] A method for enhancing corneal wound healing in a subject in need thereof, comprising contacting the ocular surface of the subject with a composition comprising a lacritin polypeptide having the sequence of SEQ ID NO: 1 or a biologically active fragment thereof. [Section 13] The ocular surface of the subject identified in step a) is KQFIENGSEFAQKLLKKFS(SEQ ID NO:5); KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7); KQFIENGSEFANKLLKKFS (SEQ ID NO: 6); and KQFIENGSEFANKLLKKFSLLKPWA (SEQ ID NO: 8), 13. The method according to claim 12, wherein the antibody is contacted with a biologically active fragment of lacritin selected from the group consisting of: or a derivative thereof which differs from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 by one or two amino acid substitutions. [Section 14] 14. The method according to item 13, wherein the amino acid substitutions are at positions 4, 6, 8, 10, 17 and 19 relative to the numbering of SEQ ID NO:7. [Section 15] 14. The method according to claim 13, wherein the biologically active fragment of lacritin consists of SEQ ID NO: 7 or a derivative thereof that differs from SEQ ID NO: 7 by one or two amino acid substitutions at positions selected from 4 and / or 19 relative to the numbering of SEQ ID NO: 7. [Section 16] Item 14. The method according to item 13 above, wherein the biologically active fragment of lacritin consists of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7). [Section 17] 17. The method according to any one of items 12 to 16, wherein the subject is recovering from PRK (laser photorefractive keratectomy) or LASIK (laser in situ keratomileusis) surgery. [Section 18] A bactericidal composition comprising a C-terminal fragment of lacritin selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 or a derivative thereof that differs from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 by one or two amino acid substitutions; and a pharmaceutically acceptable carrier, said composition being suitable for topical administration to the ocular surface of a subject. [Section 19] 19. The composition according to item 18, wherein the amino acid substitutions are at positions 4, 6, 8, 10, 17 and 19 relative to the numbering of SEQ ID NO:7. [Section 20] Item 19. The composition according to item 18, wherein the C-terminal fragment of lacritin consists of KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7). [Section 21] 21. The composition according to any one of items 18 to 20, further comprising a second antibacterial agent. [Section 22] 22. The composition according to item 21, wherein the second antibacterial agent is lysozyme. [Section 23] 23. The composition according to item 22, wherein the weight ratio of lysozyme to the C-terminal fragment of lacritin is 4:1 to 3:1. [Section 24] 19. A method for treating a corneal infection, comprising contacting the cornea of ​​a subject in need thereof with the composition of paragraph 18. [Section 25] Use of a peptide consisting of the sequence KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7) in the manufacture of a medicament for the treatment of dry eye. [Example]

[0190] Example 1 Identifying Dry Eye Disease procedure Cell culture, constructs and antibodies Human corneal epithelial (HCE-T) cells were purchased from the RIKEN BioResource Center (Tsukuba, Japan) and used between passages 3 and 15. HCE-T cells were cultured and maintained in DMEM / F-12 containing 4 mg / ml insulin, 100 μg / ml EGF, 500 μg / ml cholera toxin, and 5 μl / ml DMSO. Primary human corneal epithelial cells (PCS-700-010) were purchased from ATCC (Manassas, VA) and expanded in the recommended medium.

[0191] N-terminal deletions of 45, 65, and 71 amino acids, as well as point mutants V69S, I73S, I98S, F104S, L108S, L109S, F112S, I68S / I73S, V91S / L109S, and L108S / L109S / F112S, were developed from pLAC. All constructs were confirmed by DNA sequencing. Lacritin and deletions or point mutants (including the deletion mutant "C-25") were generated in Escherichia coli and purified as previously described (Wang et al., (2006) J. Cell Biol. 174, 689-700) and further purified against DEAR in PBS, with lacritin collected in the flow-through. Purified lacritin was filter-sterilized and stored lyophilized.

[0192] Polyclonal N- and C-terminal specific anti-lacritin antibodies were raised and differentiated in New Zealand White rabbits against keyhole limpet hemocyanin-conjugated EDASSDSTGADPAQEAGTS ("Pep Lac N-term") as "anti-Pep Lac N-term" and against lacritin-deficient mutant N-65 as "anti-N-65 Lac C-term" (Bio-Synthesis Inc., Lewisville, TX). Monoclonal N-terminal specific anti-lacritin antibodies were raised in mice against keyhole limpet hemocyanin-conjugated DPAQEAGTSKPNEEIS (University of Virginia Lymphocyte Culture Center) and tested by three rounds of cloning against lacritin-deficient mutant C-59 as 1F5-C9-F4 ("1F5"; IgG1).

[0193] Tear and viability analysis Tear fluid was collected from 0.5% proparacaine-anesthetized eyes by inserting a filter-wicking "Schirmer" test paper with millimeter gradation between the eyelid and the eye and stored individually at -70°C. Before elution, the total volume of normal or dry tears was estimated from the millimeters of tear fluid drawn onto each test paper. This defined the final volume of PBS used for elution, respectively. Pooled normal tears or pooled dry tears were stored at -70°C until use. For FOXO3 translocation assays, HCE-T cells were cultured in triplicate on glass coverslips in α-MEM (5.54 m m glucose) to subconfluence (approximately 50%), sensitized overnight in IFN-γ (100 units / ml; Roche Applied Science), and treated for 15 min with normal or dry eye tear fluid diluted 1:100 in α-MEM (50 ng / ml; PeproTech, Rocky Hill, NJ) supplemented with TNF with or without 10 nM lacritin or C-25. Cells were washed, fixed with 4% paraformaldehyde, and immunostained for FOXO3 (1:200; Millipore, Billerica, MA), followed by goat anti-rabbit secondary antibody, and viewed under a Zeiss LSM 700 microscope.

[0194] Several experiments were performed with normal tears immunodepleted for lacritin. For immunodepletion, rabbit anti-Pep Lac N-terminal and anti-N-65 Lac C-terminal were co-immobilized on protein A beads and washed. A rabbit preimmune column was prepared similarly to the mock-depleted tears. The flow-through from each overnight incubation with normal tears was collected and assayed in triplicate on TNF-IFNG-sensitized cells as described above. For validation, the acid eluate from each column was separated by SDS-PAGE, transferred to nitrocellulose, and blotted for lacritin using mouse anti-lacritin antibody 1F5 and a mouse-specific peroxidase-labeled secondary antibody, followed by chemiluminescence detection.

[0195] Viability was monitored using a 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Invitrogen) or a Nucleocounter (New Brunswick Scientific, Edison, NJ). Cells were plated at 500 cells / mm 2Cells were seeded overnight in 24-well plates at a density of 100 μg / ml and allowed to grow to approximately 80% confluence the next day. Cells were then sensitized with IFNG (100 units / ml) in α-MEM overnight and treated with 10 nm lacritin or lacritin deletion or point mutant forms, or various doses of lacritin in α-MEM plus TNF as described above, in triplicate for 15 min.

[0196] Inhibitors were included simultaneously with the addition of lacritin or C-25 in all viability and other experiments, unless otherwise noted. Inhibitors included PI103 (0.5 μM; EMD, Darmstadt, Germany), rapamycin (10 and 100 nm; EMD), and cyclosporin A (0.1 μM; EMD). One exception was 4-methylumbelliferyl-β-d-xylopyranoside ("xyloside"; 70 and 80 nm; Sigma), which was added during IFNG intercropping and TNF and lacritin treatment. Assays were completed by the addition of MTT (5 mg / ml) to each well, followed by isopropanol with 0.04 N HCl (4 h at 37°C) and measured at 570 nm with a reference wavelength of 630 nm. Viability was assayed in a Nucleocounter (New Brunswick Scientific).

[0197] result Tears accumulate on the avascular membranous epithelium and vascular conjunctiva as a translucent film rich in proteins, lipids, and metabolites. Beyond their ability to moisturize the eyelids, tears are essential for the refraction of light. The role of tears is equally important in promoting corneal epithelial health and cannot be replaced by drugs or eye drops. When tears are chronically insufficient, the epithelium becomes stressed and releases proinflammatory cytokines, further exacerbating the situation. Dry eye affects 5-6% of the general population, increasing to 6-9.8% and 34% in postmenopausal women and the elderly, respectively. Despite being the most common eye disorder, there is no single gold-standard diagnostic test and no effective treatment. Current methods include: a) subject questionnaires, b) rose bengal or lissamine green staining for ocular surface damage, c) Schirmer dipstick measurement of tear volume, d) tear breakup time, e) tear evaporation rate, f) tear meniscus height or half-system, g) tear film index or turnover rate, h) tear osmolality, i) lysozyme or lactoferrin assays, and j) tear fern analysis, each of which has numerous drawbacks.

[0198] The tear proteome is estimated to contain 1,543 proteins, more than half of which are designated as "intracellular" by gene ontology, suggesting that cell death from normal epithelial regeneration may contribute. The only growth factor-like molecule downregulated from mild to severe water deficiency was lacritin. Comparison of tears from 73 normal individuals with 129 individuals with aqueous-deficient dry eye by 2-D SDS-PAGE revealed that lacritin was downregulated in 95% of cases. When applied topically in rabbits, lacritin promoted basal tearing. Another tear protein found to be downregulated in dry eye was lipocalin-1. Lipocalin-1 cleanses the ocular surface of lipids that would otherwise prevent it from moistening. Lacritin was the protein most severely downregulated in contact lens-associated dry eye, likely due in part to its easy adsorption onto contact lenses. It is also a common inflammation of the eyelids and is deficient in blepharitis associated with evaporative dry eye. However, 2-D SDS-PAGE before mass spectrometry analysis is required to identify lacritin downregulation, making the method impractical for clinical use.

[0199] Several molecules are required for lacritin activity. An unusually deglycanated form of syndecan-1 (SDC1) was discovered to be the major cell surface binding protein for lacritin by mass spectrometry sequencing of cell surface proteins bound to a lacritin column in physiological salts. Validation was by affinity precipitation. SDC1 is a widely expressed cell surface heparin sulfate proteoglycan with a carboxy terminus anchored to the plasma membrane by a short cytoplasmic tail, and an ectodomain with or without short chondroitin sulfate chains, proximal chondroitin sulfate chains at serines 184 and 194 (human SDC1; numbering excludes the signal peptide), and up to three distal heparin sulfate chains (serines 15, 23, and 25). The C-terminal α-helix of lacritin connects domains within amino acids 1–50 of SDC1, and binding is dependent on prior heparanase deglycanization of the heparin sulfate. SiRNA knockdown of SDC1 abrogates lacritin-dependent mitogenic activity, resulting in heparanase (but not heparanase-2) depletion, but can be rescued by the addition of exogenous heparanase or bacterial heparitinase. The binding domain is narrowed to the hydrophobic amino acids 20–30 that enhance lacritin's C-terminal α-helicity. Binding also depends on the substitution of S23 and S25 (potentially S15) with heparin sulfate and chondroitin sulfate, a novel hybrid domain of heparanase-cleaved heparin sulfate and adjacent chondroitin sulfate hydrophobic core proteins. Heparanase is not widely expressed. N-terminal substitution of SDC1 with chondroitin sulfate is not common.

[0200] We used a highly sensitive fluorescence assay to examine SDC1 in tears. Tear fluid from 146 individuals undergoing vision-correcting laser photorefractive keratectomy or LASIK surgery was collected on Schirmer strips, with additional tear fluid collected 1 day, 1 week, and 1 month later. Tears were stored at -70°C, eluted with an equal volume of PBS, pooled by time and by normal (≥15 mm) versus dry eye (≤5 mm) tears, and then separated by SDS-PAGE. Separated tear proteins were transferred to nitrocellulose and blotted with anti-SDC1 monoclonal antibody A-38B. Secondary Abs were precleared for the tear column, and Ab C-terminally for the C-59 lacritin truncation mutant. Normal tears were unexpectedly enriched in rare heparanase-deglycanized forms of SDC1 targeted by lacritin (Figure 1). Most deglycanized forms of SDC1 were absent in dry eye tears. Deglycanized SDC1 can vary in molecular weight from approximately 90 kDa (Figure 1) to approximately 80 kDa or even approximately 60 kDa, depending on the level of O-glycosylation, which can vary among various epithelia. One day after laser photorefractive keratectomy or LASIK surgery, tear fluid approximately 90 kDa SDC1 was indistinguishable between normal and dry eye, consistent with surgery-induced dry eye. A novel approximately 25 kDa SDC1 fragment was observed in what was initially designated dry eye (Figure 1). Deglycanized SDC1 and tearing recovered in normal individuals 1 week and 1 month later. However, the dry eye-associated approximately 25 kDa band remained throughout the assayed time frame (Figure 1). Thus, approximately 90 kDa syndecan in tears is a marker of normality. Those lacking the approximately 90 kDa syndecan have dry eye. The approximately 25 kDa form also identifies individuals with dry eye who have undergone PRK or LASIK.

[0201] Blotting for the inactive lacritin-C splice variant in tears was also found to be indicative of dry eye (Figure 2A). Lacritin-C lacks sequences from exons 4 and 5, which encode the C-terminus, and contains additional sequences not present in native lacritin. Instead, a new C-terminus of the inactive form is spliced ​​out from intron 3. We further found differences in tear heparanase, with latent heparanase being more abundant in normal tears (except 1 day after laser photorefractive keratectomy or LASIK surgery; Figure 3), whereas active heparanase was more abundant in dry eye tears. Secretion of heparanase, which is processed from its latent 65 kDa form to its active 58 kDa heterodimer form, is stimulated by UTP. UTP is a proposed treatment for dry eye through a mechanism thought to involve mucin production. These observations suggest a potential link between physiological lacritin, UTP, and heparanase on the ocular surface.

[0202] In summary, tears in aqueous-deficient dry eye are significantly less likely to contain deglycanized SDC1 and latent heparanase, but more likely to contain SDC1 fragments, chronically active heparanase, and inactive lacritin-C splice variants with normal active lacritin. These conditions are suitable for the exacerbation or onset of dry eye, which can be reversed by topical restoration of lacritin.

[0203] Identifying Dry Eye Disease - Specific Treatment Commonly used "artificial tears" temporarily relieve symptoms associated with dry eye without addressing the cause of these symptoms. Ophthalmic formulations of the anti-inflammatory drug cyclosporine are currently widely used. It and other anti-inflammatory agents have been clinically tested, but generally benefit only approximately 15% of dry eye patients. Rather than focusing on the consequences of inflammation or applying drugs developed for other organ systems, it is beneficial to consider the natural biology of the ocular surface and what is at fault in dry eye. Downregulation of a native tear protein called lacritin monomer, which promotes basal tearing when applied topically to normal rabbit eyes, may be an upstream instigator of dry eye disease. Why is lacritin monomer so scarce in dry eye? Lacritin monomer is crosslinked to inactive multimers by tissue transglutaminase (TGM2) in tears. This was demonstrated by immunodepleting all lacritin monomers, multimers, and fragments from human tears. Recombinant lacritin spiked into immunodepleted tears formed dimers, trimers, and tetramers after overnight incubation at 37°C. A small amount of dimers was formed in the negative control without tears. The crosslinker contains glutamine 106 within the lacritin mitogenic domain (amino acids 100–109), which targets syndecan-1. Crosslinked lacritin binds substantially less to syndecan-1 and is less active (Figure 5; right two bars). Blotting suggests that normal human tears contain 0.6 μM TGM2, which therefore appears to act as a negative regulator of monomeric lacritin. Human corneal epithelial cells express both TGM1 and TGM2 mRNA. Expression of both mRNAs increases with hyperosmotic stress, particularly TGM1, but TGM1 was not detected in tears. Thus, lacritin may undergo enhanced crosslinking and deactivation in dry eye.

[0204] Truncation and point mutants were generated to define the lacritin domain required for regulating homeostasis. The inactivity of the C-25 truncation mutant was still shown to be α-helical and likely amphipathic, thus defining a cytoprotective domain at the C-terminus of lacritin. The hydrophobic face of the amphipathic α-helix may mediate high-affinity agonist-receptor or coreceptor interactions. To assess this possibility, hydrophobic residues were mutated one-, two-, or three-fold. We also generated truncated and C-terminal lacritin-C splice variants ("I3") with sequences completely different from those of the wild-type. Amino acid numbering throughout is that of the mature protein without the signal peptide. The hydrophobic face mutants I98S, F104S, L108S / L109S / F112S, and F112S were significantly less active (Wang et al., 2013). Activity was unaffected by mutations L65S, I68S / I78S, V69S, and I73S in the adjacent α-helix. Deletion of 45, 65, or 71 N-terminal amino acids was ineffective, and I3 was inactive. L108, L109, and F112 interact with the syndecan-1 core protein sequence GAGAL.

[0205] Basal tears from normal individuals or those diagnosed with dry eye were incubated with human corneal epithelial cells stressed with the inflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor (TNF) (more similar to in vivo dry eye equivalents). Nuclear-cytoplasmic translocation of the corneal transcription factor FOXO3 served as a simple readout for cellular stress, with cytoplasmic FOXO3 indicating restored homeostasis. Nuclear FOXO3 is generally translocated upon cellular stress or death. In stressed cells treated with normal tears, FOXO3 translocated to the cytoplasm. However, in dry eye tears, FOXO3 remained nuclear. Next, lacritin was immunodepleted from normal tears, although normal tears have other growth factors that may complement it. Dry eye tears were also spiked with lacritin. Dry eye tears are rich in both hyperosmolar and inflammatory cytokines. Simulated tear fluid translocated FOXO3 to the cytoplasm, whereas FOXO3 remained nuclear in cells treated with lacritin-depleted tears. Dry eye tears spiked with lacritin, but not with the lacritin truncation mutant C-25 (lacking the C-terminal 25 amino acids), translocated FOXO3 to the cytoplasm. Lacritin, but not C-25, also translocated FOXO3 in primary human corneal epithelial cells subjected to IFN-γ / TNF stress. Thus, lacritin is a potent protector of normal tears.

[0206] This study was repeated using the C-terminal fragment of bioactive lacritin (LACRIPEP; SEQ ID NO: 7). Cultured human corneal epithelial cells were treated with inflammatory cytokines to induce stress as described above, and the cells were then treated with 10 nM of the inactive lacritin truncation mutant (C-25), lacritin, or LACRIPEP. Measurement of cytoplasmic staining in the FOXO3 assay (nuclear FOXO3 staining indicates cell death) indicates that LACRIPEP is equally active as lacritin (see Figure 4A) in enhancing cell survival compared to the negative control (C-25). Therefore, we anticipate that LACRIPEP may be a viable alternative to lacritin for all applications.

[0207] Autoimmune regulator (Aire)-deficiency [Aire - / - ] Mice spontaneously develop dry eye without the need for a dry chamber or sporadic administration. - / - Mice received 10 μl of 50 μg / ml lacritin or PBS (control) three times daily for 3 weeks. Several different assays monitored the results. A bioactive fragment of lacritin, LACRIPEP (SEQ ID NO: 7), prevented tear loss and reduced lacrimal gland inflammation, as dry eye disease developed in Aire(- / -) dry eye mice (Figure 4B; filled circles) compared with those receiving topical PBS (open circles). Topical lacritin reduced CD4+ T-cell infiltration into the lacrimal gland, measured as the number of lymphocytic foci per square millimeter of lacrimal tissue (3.68±0.65 lacritin per square millimeter vs. 9.7±1.5 PBS per square millimeter; P=0.01), but had no apparent effect on the pattern or distribution of CD4+ T cells in either the corneal stroma (14.6±1.6 lacritin vs. 12.4±2.1 PBS) or limbus (29.6±2.5 lacritin vs. 34.6±2.9 PBS).

[0208] To assess ocular surface mucosal damage from dry eye, topical application of lissamine green to the eyes of Aire(- / -) dry eye mice progressively stained PBS-treated eyes over time (see Figure 4C). In contrast, topical lacritin significantly reduced staining (-0.417 ± 0.06 for lacritin vs. 0.125 ± 0.07 for PBS; p = 0.02). Furthermore, lacritin reduced keratin 10 (a skin epithelial marker) levels, suggesting its ability to prevent corneal keratinization associated with chronic inflammation, while keratin 12 (a corneal marker) expression remained stable (80.1 ± 4.8% for lacritin vs. 85.6 ± 1.8%; P > 0.10). Furthermore, Aire(- / -) dry eye mice treated with lacritin also showed less corneal staining, an indicator of cell death, as they developed dry eye disease (Figure 4C; filled circles) compared with PBS (open circles). Thus, topical lacritin and its bioactive fragments reduced lacrimal gland inflammation and corneal staining and promoted ocular surface differentiation in dry eye. Importantly, the suppression of inflammation and promotion of tear flow were achieved without direct contact with either inflammatory or tear-producing cells.

[0209] Topical lacritin stimulates tearing without physical access to lacrimal acinar cells. The rapidity of the response is consistent with corneal sensory nerve activation. Individual corneal sensory nerve activity was monitored at the level of the trigeminal ganglion in rats using previously described methods. These studies revealed that topical lacritin is neuroactive. Topical lacritin enhanced the nerve "dryness response" and, to a lesser extent, the nerve "wetness response." The "dryness response" refers to nerve activation as a result of corneal drying, which is thought to be a TRPM8-mediated stimulus important for tearing, whereas the "wetness response" occurs when an agonist is present in the corneal nerve terminals. Negative control truncation mutant C-25 did not affect either of these responses, supporting the importance of the C-terminal α-helix in both nerve stimulation and tearing. The enhancement of the dryness response by lacritin is likely due to modulation of the TRPM8 channel: adrenergic α-helix. 2A and / or α 2CThe receptors range from a fully inhibited TRPM8 state during corneal hydration (with lacritin on board) to a fully reduced (activated) state during corneal dryness (with lacritin removed). Because TRPM8 activity is initially low during corneal hydration, the apparent inhibition of action potentials by lacritin during corneal hydration is small, whereas it reaches an optimal level during corneal dryness when dynamic cooling of the ocular surface occurs. Lacritin can also increase TRPM8 neuronal density.

[0210] Stimulation of the dryness response could have been achieved by indirect or direct mechanisms. Lacritin stimulation of the corneal epithelium could indirectly target sensory neurons via junction-like complexes between the two cell types. However, these are considered rare. Epithelial tight junctions that prevent lacritin access to nerve endings argue against a direct mechanism. However, Ca 2+ Some growth factors can loosen tight junctions. PDGF permeabilizes tight junctions between cultured kidney cells within minutes, as does VEGF for endothelial tight junctions, while chronic permeabilization of surface cells of stratified corneal epithelium is observed in MMP9- or inflammatory cytokine-associated inflammation and bacterial infection from endotoxin challenge. Lacritin-dependent Ca 2+ This recruitment may be sufficient to promote rapid, acute permeabilization for neural access. We predict a two-step process. First, because calcium regulates tight junction permeability, targeting lacritin or lacritin peptides to superficial corneal epithelial cells promotes subtle loosening of tight junctions, possibly through a transient increase in occludin trafficking to early endosomes or lacritin-dependent calcium signaling in the corneal epithelium. We predict that the process is activated within 1 minute, given that lacritin-stimulated calcium signaling occurs within 20 seconds and lacritin-stimulated autophagy occurs within 1 minute. Thus, lacritin or peptides enter the corneal epithelium. Subsequent neural stimulation may be sufficient to trigger tight junction reclosure, as per the importance of neural stimulation in corneal wound healing.

[0211] Syndecan-1 is a cell surface heparin sulfate proteoglycan that mediates cellular lacritin targeting, but only after heparanase (Ma et al., '06) did it express GAGAL, ​​which is located between the heparin sulfate chains. Heparanase also generates residual heparin sulfate that appears to be required for lacritin binding, suggesting a hybrid GAGAL / heparin sulfate binding site. To assess the role of this interaction, cells were incubated overnight in 4-methylumbelliferyl-bD-xylopyranoside ("xyloside") to competitively inhibit heparan and chondroitin sulfate assembly. Xyloside completely abolished the cytoprotective activity of lacritin. Thus, these activities depend on a region in the C-terminus that contains the syndecan-1 binding domain. Furthermore, lacritin activity appears to be fully integrated within the sequence KQFIENGSEFAQKLLKKFS (“N-94 / C-6”; SEQ ID NO: 5) (Wang et al., 2006) or KQFIENGSEFAQKLLKKFSLLKPWA (“N-94”; SEQ ID NO: 7) (Zhang et al., 2013), which is as potent as lacritin when produced synthetically.

[0212] Lacritin targeting of corneal sensory neurons. Adrenaline α 2C The selective antagonist MK912 inhibits lacritin-accelerated autophagy in HCE-T cells, as does the syndecan-1 inhibitor xyloside. Both also inhibit lacritin-stimulated FOXO3 phosphorylation. Activity profiles of corneal neurons before, during, and 1 hour after 10 μM lacritin reveal that a small inhibition (approximately 12 to 8 spikes / s) follows immediately after lacritin application, likely due to α2-adrenergic receptor activation, which inhibits TRPM8 channels. Removal of this inhibition and washout 1 hour after lacritin results in enhanced excitation of dry (approximately 20 to 23 spikes / s) and wet responses (approximately 12 to 16 spikes / s).

[0213] Example 2 The monomeric form of tear lacritin is a multifunctional factor involved in the alleviation of ocular surface stress. It is also an agonist of basal tearing. Monomeric lacritin targets the heparanase (HPSE)-deglycanated form of syndecan-1 (SDC1) on the cell surface. However, both polymerized lacritin and the lacritin-C splice variant are unable to target SDC1 and are therefore inactive. We investigated whether either SDC1 or HPSE can replace monomeric lacritin in tears in dry eye, and whether SDC1 or HPSE may be inappropriate.

[0214] method: Tear fluid was collected on Schirmer strips from 146 individuals before and 1 day, 1 week, and 1 month after laser photorefractive keratectomy. Tear fluid was stored at -70°C and subsequently eluted with an equal volume of PBS. The tears were pooled by time and by normal (≥15 mm) versus dry eye (≤5 mm) tears. Tear fluid was separated by SDS-PAGE and blotted with anti-N-terminal specific lacritin mab 1F5, anti-C-terminal specific lacritin ab "ab C-terminal," anti-lacritin-C splice variant mab 4G6, anti-SDC1 mab A-38B, and anti-heparanase abs #733 and #1453. Secondary abs were precleared for the tear column, and ab C-terminal was precleared for the C-59 lacritin truncation variant.

[0215] result: Ab C-terminus detected less lacritin monomer in dry eye versus normal tears, and the deficiency was apparently compensated for in dry eye by an increase in the lacritin-C splice variant. The 1F5 mab epitope is shared by both forms, and therefore the putative hybrid band is more prevalent in dry eye. Normal tears were enriched in uncleaved HPSE and deglycanated SDC1. One day after PRK, lacritin-C further increased in dry eyes, whereas both SDC1 and HPSE decreased in normals. Return to the pre-PRK state was evident by one month.

[0216] Conclusion: Aqueous-deficient dry eye tears are associated with decreased lacritin monomer, increased lacritin-C splice variants, and less deglycanized SDC1 and latent HPSE, conditions that are appropriate for the worsening or initiation of dry eye disease.

[0217] Example 3 Stability of the C-terminal fragment of the 25-amino acid lacritin. A limitation of most synthetic peptide drugs is their protease sensitivity. According to PROSPER (Protease Specificity Prediction Server) analysis, only the HIV proteases retropepsin and cathepsin K can cleave LACRIPEP, the former cleaving in the middle and the latter removing the final alanine. Retropepsin would inactivate LACRIPEP, whereas cathepsin K would have no effect. However, neither protease is found in normal human tears. Nevertheless, tears are rich in other proteases. Therefore, we incubated LACRIPEP in normal human tears at 37°C for 2, 4, 6, and 16 hours. For immunoblotting, we first removed all endogenous lacritin by immunodepletion. As shown in Figure 6A, which shows immunoblots of the protease-sensitive positive control "SN pep" and LACRIPEP ("N-94") from various proteins after incubation in lacritin-depleted human tear fluid for 2 to 16 hours at 37°C, LACRIPEP was highly stable for at least 16 hours. Mass spectrometry analysis of SN pep, Lacripep ("N-94"), and Lacripep lacking the six C-terminal amino acids ("N-94 / C-6") demonstrates the comparative stability of the three peptides after incubation in lacritin-depleted tear fluid for 4 hours at 37°C (Figure 6B). Surprisingly, the smaller C-terminal fragment of lacritin (N-94 / C-6; SEQ ID NO: 5) was found to be less stable than the LACRIPEP peptide (SEQ ID NO: 7). Mass spectrometry analysis suggested that Lacripep ("N-94") and Lacripep lacking the six C-terminal amino acids ("N-94 / C-6") had similar stability in tears, and that Lacripep lacking the six C-terminal amino acids ("N-94 / C-6") was stable in phosphate-buffered saline for 29 days at 62°C (Figure 6B). However, immunoblotting showed that N-94 / C-6, but not Lacripep ("N-94"), consumed the epitope after incubation in lacritin-depleted tears for 4 hours at 37°C.Thus, the last six amino acids of native lacritin are relevant to increasing the stability of the biologically active fragment of lacritin making N-94 a superior pharmaceutical peptide compared to N-94 / C-6.

[0218] Another advantage of LACRIPEP is its low optimal dose. In human cell cultures, its optimal dose is 1-10 nM. In animal studies, approximately 4 μM (0.0012%) is optimal (Figures 7A and 7B). 4 μM LACRIPEP was also found to be bactericidal but not hemolytic. As a whole protein, lacritin has no bactericidal activity.

[0219] To monitor lacripep in systemic toxicity studies, lacripep was synthesized with a single C-terminal tyrosine for iodination. 125 I-Lacripep-Y was formed. A single 4 μM dose 125 Rats administered I-Lacripep-Y showed high retention in ocular tears with minimal levels detected in blood and serum (see Figure 8).

[0220] Example 4 Cleavage-enhancing fragments of tear lacritin are bactericidal Experimental procedure Tears and Tear Immunodepletion: Basal tears from normal humans were collected. Briefly, tears from eyes anesthetized with 0.5% proparacaine were collected into pre-weighed cores and flash-frozen for storage at -70°C. Tears were eluted by immersing each test strip in 30 μl of PBS for 20 minutes, followed by centrifugation. For immunodepletion, 10-fold diluted tears were incubated overnight (4°C) or for 1 hour at room temperature with protein A beads (0.2 ml, NAb Spin Kit, Peirce / Thermo Scientific) saturated with anti-N-65 Lac C-terminus or preimmune Ig. N-65 is a truncated lacritin variant lacking the 65 N-terminal amino acids. After centrifugation (5000 × g for 1 minute), tear flow-through was then assayed for antibacterial activity.

[0221] Lacritin constructs, purification, synthetic peptides, and mass spectrometry. Lacritin N-terminal truncations N-55, N-65, N-71, and N-75 were produced by PCR from the parent cDNA pLAC as previously described (Zhang et al., (2013) J. Biol. Chem. 288, 12090-12101). N-terminal deletions of 80 (N-80) and 86 (N-86) amino acids were amplified using forward primers AAG1-A ... GGTGGTCATATGAAAGCAGGAAAAGGAATGCACGG (SEQ ID NO: 9) and GGTGGTCATATGCACGGAGGCGTGCCAGGTGG (SEQ ID NO: 10) and the common reverse primer GGTGGTCATATGTATATCTCCTTCTTAAAG (SEQ ID NO: 11). All constructs were verified by sequencing. Bacterial protein expression and purification of recombinant lacritin and lacritin truncation were performed as previously described (Zhang et al., (2013) J. Biol. Chem. 288, 12090-12101). Briefly, clarified cell (ER2566 or BL21-CP) lysates were loaded onto a chitin column (IMPACT-CN System; New England Biolabs Inc., Beverly, MA) equilibrated with 50 mM Tris, 0.5 M NaCl (pH 8), followed by 20 column volumes of washing and elution with 50 mM 2-mercaptoethanol for 16 hours at room temperature. Extensive dialysis against PBS (4°C) was performed, followed by protein quantification. Further DEAE purification removed approximately 9-kDa lacritin proteolytic fragments and bacterial contaminants, in which lacritin was collected as flow-through using 140 mM NaCl in phosphate buffer, pH 7.2. Synthetic peptides N-80 / C-25, N-94, N-94 / C-6, N-94 / C-10, N-94 / C-15, N-99, and N-104 were synthesized by Genscript (Piscataway, NJ) with acetylated N-termini. Purity was 95%. All C-termini except lacritin C-termini N-94, N-99, and N-104 were amidated. N-64 / C-31 was neither amidated nor acetylated and was synthesized by the University of Virginia Biomolecular Research Facility. The nature of the approximately 9-kDa lacritin fragment was examined by Western blot. Briefly, lacritin before and after DEAE was separated by SDS-PAGE, then transferred and blotted with anti-Pep Lac N-terminal and anti-N-65 Lac C-terminal antibodies, respectively, diluted 1:200 or 1:400 in PBS containing 0.3% Tween 20. Detection was by ECL.

[0222] For fragment purification, chitin-enriched lacritin was dialyzed against phosphate buffer (pH 7.2) containing 14 mM NaCl. After incubation with DEAE equilibrated with the same buffer, the approximately 9-kDa fragment was collected in the flow-through, while intact (18 kDa) lacritin was eluted with 140 mM NaCl in phosphate buffer, pH 7.2. After protein concentration determination (BCA assay), both fractions were aliquoted, lyophilized, and stored at -70°C. Analysis was performed by SDS-PAGE on a 4-20% gradient gel. The identity of the approximately 9-kDa fragment was determined by mass spectrometry.

[0223] Bacterial growth, SYTOX Green assay, and on-column cleavage—E. coli (ATCC (Manassas, VA) catalog no. 10536), S. epidermidis (ATCC catalog no. 12228), and P. aeruginosa (ATCC catalog no. 9027) were grown to mid-logarithmic phase in 50 ml of Luria-Bertani (LB) medium and washed three times by centrifugation with phosphate buffer (pH 7.2) containing 10 mM NaCl (PB-NaCl). The pellet was resuspended in 1 ml of PB-NaCl.

[0224] For lacritin inhibition assays, 50 μl of bacterial pellets, each diluted 1:100 in PB-NaCl, were incubated for 1.5 hours (37°C) with 100 μl of lacritin, lacritin truncations, or synthetic peptides at final concentrations of 0.1–6 μM. The mixtures were diluted 1:10 in PB-NaCl, and 100 μl were then plated in quadruplicate on LB agar plates and grown overnight at 37°C. Colonies were counted manually. In another example, mid-logarithmic E. coli were treated with 2 μM lacritin or lacritin truncations, or ampicillin (5 μM), or tetracycline (2 μM) for 0, 1, 2, or 3 hours at 37°C. After each treatment, 100 μl was centrifuged, resuspended in 1 ml of PB-NaCl, plated on LB agar (100 μl), grown overnight (37°C), and colonies were counted.

[0225] For salt sensitivity testing, pelleted and washed mid-log E. coli, S. epidermidis, or P. aeruginosa were resuspended in 1 ml of PB-NaCl and treated with 3 μM N-65 in PB-NaCl or 130, 280, or 380 mosmol / liter PB-NaCl as described above for 1.5 hours at 37°C. The mixtures were diluted 1:10 in PB-NaCl, after which 100 μl of each was plated in quadruplicate on LB agar plates and grown overnight at 37°C. Colonies were counted manually.

[0226] For bacterial permeability assays, pelleted and washed mid-logarithmic E. coli cells were resuspended in 1 ml of PB-NaCl and treated with 3 μM lacritin, N-65, or C-25 or 10% Triton X-100 as described above. Similarly, washed mid-logarithmic S. epidermidis cells were resuspended in 1 ml of PB-NaCl and treated with lacritin, C-25, or the approximately 9-kDa purified lacritin fragment. Then, 1 μL of 0.5 mM SYTOX Green was added to each well of a 96-well fluorescence microtiter plate. Readings were taken at 5-minute intervals at excitation and emission wavelengths of 485 nm and 538 nm using a Fluoroskan Ascent FL fluorometer (Thermo Fisher Scientific). In parallel, SYTOX Green internalization was visualized by confocal microscopy after 1 hour of treatment of washed mid-log E. coli with 10% Triton X-100, PB-NaCl, or 3 uM N-65.

[0227] For cell-free synthesis without glycosylation, the full-length lacritin cDNA in pLacSL was PCR amplified and subcloned into pTXB1, provided by the manufacturer (New England Biolabs, Ipswich, MA). Cell-free synthesis and subsequent ribosome removal followed by metal affinity resin adsorption of the His-tagged factor were performed according to the manufacturer's instructions (New England Biolabs; PURExpress). Immediately after expression, aliquots were stored at -60°C. Other aliquots were incubated at 37°C for 24 and 48 hours. Each was separated by SDS-PAGE, transferred, and blotted with an anti-N-65 Lac C-terminal antibody.

[0228] For lacritin cleavage assays, supernatants from saturated 50-ml overnight cultures of S. epidermidis were collected by centrifugation (10 min; 11,000 rpm). Each supernatant was then incubated with N-terminally immobilized lacritin-intein-containing chitin beads in PB-NaCl for 4, 16, and 20 hours (37°C). C-terminal cleavage products were collected by PBNaCl washes, separated by SDS-PAGE, transferred, and blotted with anti-N-65 Lac C-terminal antibody. In some experiments, supernatants and lysates from overnight cultures of S. epidermidis, S. aureus, P. aeruginosa, and E. coli were incubated overnight (37°C) with lacritin in PB-NaCl solution. The mixtures were then separated by SDS-PAGE, transferred, and blotted with anti-N-65 Lac C-terminal antibody. Parallel studies monitored the integrity of chitin-intein-immobilized lacritin in PB-NaCl at 37°C for 0, 24, 48, and 72 hours or for 24 hours (37°C) with 1 μM pepstatin, 10 μM bestatin, 100 μM antipain, 1 mM 4-benzenesulfonyl fluoride hydrochloride, 100 μM chymostatin, 10 μM E64, 100 μM leupeptin, or 10 mM phosphoramidon or after boiling at 100°C for 5 minutes.

[0229] Hemolysis assay—The method of Cerovsky et al. was followed with some modifications. Washed sheep red blood cell pellets (MP Biomedicals, Santa Ana, CA) were suspended in 565 μl of PBS plus 100 μl of lacritin, N-55, N-65, N-71, N-75, N-80, or C-25 at a final concentration of 2 μM, or N-65, N-64 / C-31, N-80 / C-25, N-94, N-94 / C-6, N-94 / C-10, N-94 / C-15, N-99, or N-104 at a final concentration of 6 μM for 1 hour at 37°C. Triton X-100 (5% final concentration) or PBS was included instead of lacritin or lacritin fragments as positive and negative controls, respectively. After centrifugation (250 xg; 5 min), the absorbance of the supernatant was monitored at 540 nm.

[0230] Metabolomic analysis - Washed mid-logarithmic E. coli cells were incubated with 6 μM N-65 or PB-NaCl for 15 min at 37°C in six replicates, each containing 1 × 10 8 The cells were incubated with 1000 mg of 10 ...

[0231] Statistical Analysis—All experiments, except for single-metabolome analyses, were performed at least three times. Statistical analysis of metabolite data was performed, in which raw data values ​​were first log-transformed to ensure a close normal distribution, and then evaluated with nonparametric Wilcoxon and two-sample t-tests. For both tests, p < 0.05, metabolites were considered significantly different and further analyzed for their correlation patterns by hierarchical clustering. Data are reported as mean + / - SE.

[0232] result Lacritin Bactericidal Activity in Tears—Tears protect the ocular surface from environmental pathogens and are rich in the secretagogue-promoting mitogen lacritin, which flows over the eye during basal and reflex tearing. Lacritin is 21% identical to dermcidin, whose proteolytically processed C-terminus contributes to the bactericidal activity of human sweat. We sought to determine whether lacritin or lacritin fragments possess bactericidal activity. Semidiluted basal tears completely blocked Escherichia coli growth, which, like Pseudomonas aeruginosa, is a significant contributor to bacterial conjunctivitis in developing countries. We tested both lacritin and C-terminal lacritin fragments in tears that had been immunodepleted with immobilized anti-N-65 Lac C-terminal antibody (ab C-term) or preimmune Ig (sham depletion). Both were diluted 10-fold for dose-dependent challenge with E. coli and P. aeruginosa. Sham-depleted tears suppressed E. coli and P. aeruginosa colonies in a tear volume-dependent manner, in contrast to C-terminal antibody-immunodepleted tears, which were ineffective as a phosphate buffer negative control.

[0233] The C-terminus of lacritin contains a bactericidal domain. The C-terminus of lacritin contains three predicted α-helices, each of which has been confirmed by circular dichroism. The most C-terminal α-helix is ​​amphipathic, with mostly hydrophobic surface residues, targeting syndecan-1, an initiator of corneal epithelial cell proliferation and survival. The binding of the amphipathic α-helix to bacterial membranes can be destabilizing. To explore whether these or other lacritin domains are bactericidal, we produced recombinant lacritin and lacritin truncations. Each was produced as an intein fusion protein and purified with chitin. The intein tag was removed and purified with DEAE to remove bacterial contaminants. Equimolar (2 μM) amounts of lacritin and truncations were then assayed in the presence of mid-logarithmic Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus epidermidis. Pseudomonas aeruginosa is an ocular pathogen frequently responsible for keratitis associated with contact lens wear. Staphylococcus epidermidis is a common cause of conjunctivitis and keratitis and is frequently involved in blepharitis, an eyelid inflammation associated with slightly altered tear composition, selectively containing small lacritin. Untruncate lacritin did not affect colony appearance, and colony counts were identical to those in the phosphate buffer negative control. However, colony reduction was evident with lacritin lacking 65 (N-65) or 80 (N-80) amino acids from the N-terminus, an effect that was completely or partially reversed by removal of six additional amino acids (N-86) in Escherichia coli or Pseudomonas aeruginosa, but not in S. epidermidis. Amino acids 81-86 contain the sequence LAKAGKG (SEQ ID NO: 12), which aligns with 44% amino acid identity to the sequence in the potent dermcidin fragment SSL-25.

[0234] To confirm whether the LAKAGKG (SEQ ID NO: 12) region was responsible, we generated AKAGKGMHGGVPGG (SEQ ID NO: 13; amino acids 81-94; N-80 / C-25), which contains a truncated, constricted portion of the SSL-25 homology region. We also generated the overlapping LKSIVEKSILLTEQALAKAGKGMH (SEQ ID NO: 14; amino acids 65-88; N-64 / C-31) and C-terminal KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7; amino acids 95-119; N-94). Unexpectedly, colonies were enriched for N-80 / C-25 and N-64 / C-31, while few or no colonies possessed N-94, a region that is only 12.5% ​​identical to the C-terminus of dermcidin. To narrow this region, we generated synthetic peptides with amino acids sequentially removed from the carboxy-terminus N-94 / C-6, N-94 / C-10, N-94 / C-15, and N-99 ENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 15), and N-104 (FAQKLLKKFSLLKPWA (SEQ ID NO: 16)). N-94 and N-104 were fully active, whereas the other peptides were not, although N-94 / C-6 (SEQ ID NO: 5) was slightly active. N-65 is bactericidal and equipotent to ampicillin. In dose-response studies, N-104 was nearly as effective as N-65, with half-maximal inhibition of approximately 1 μM for E. coli and approximately 1-1.5 μM for P. aeruginosa, dose ranges common for antimicrobial peptides.

[0235] Consideration The rationale for exploring whether lacritin is bactericidal was its 21% identity with dermcidin, whose proteolytically processed C-terminus contributes to its bactericidal activity in human sweat and tears. Surprisingly, dermcidin primary sequence homology was not the source of lacritin activity. Only 40.7% identity exists between the dermcidin bactericidal SSL-25 peptide and the homologous lacritin region, which was inactive as a synthetic peptide. Rather, the lacritin N-104 fragment, which shares 7% dermcidin identity, embodied the core activity. Together, this hybrid domain consists of an N-terminal amphipathic α-helix and a hydrophobic C-terminal coiled-coil tail, suitable for bacterial membrane contact and insertion, as evidenced by rapid penetration of membrane-impermeable SYTOX Green in N-65-treated cells. Surprisingly, it was found that the C-terminal 25 amino acid fragment KQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 7; amino acids 95-119; N-94) was fully active, whereas removal of the 6 terminal amino acids (e.g., KQFIENGSEFAQKLLKKFS; SEQ ID NO: 5) substantially reduced the bactericidal activity of the peptide.

[0236] Example 5 Although topical application of ophthalmic products remains the most popular and well-tolerated route of administration due to patient compliance, the bioavailability of eye drops is severely hindered by blinking, basal and reflex tear secretion, and nasolacrimal drainage. One solution to enhance the therapeutic index of topical treatments is through the application of polymeric nanoparticles as drug carriers.

[0237] One solution to enhancing the therapeutic index of localized treatments is the application of polymeric nanoparticles as drug carriers. Polymeric nanoparticles that display therapeutic ligands in the corona can interact with complex biomolecular architectures through multiple simultaneous interactions (multiplexing) and exhibit the well-defined size required for efficient tissue penetration. One such material that can be used as a scaffold is thermoresponsive elastin-like polypeptide (ELP). ELP is composed of a repeating pentapeptide motif (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 24) and exhibits a unique reversible inverse phase transition temperature, Tt, below which it becomes soluble and above which it phase separates. Tt can be tuned by choosing the guest residue (Xaa) and varying the number of five repeats, n.

[0238] Motivated to further explore the function of lacritin at the ocular surface, enhance its bioavailability, and further target it to the corneal epithelium, we utilized a diblock ELP(SI) nanoparticle scaffold to bioengineer LSI nanoparticles with multivalent display of lacritin on their surface.

[0239] material and method Materials and equipment TB DRY® Powder Growth Media was purchased from MO BIO Laboratories, Inc. (Carlsbad, CA). NHS-rhodamine was purchased from Thermo Fisher Scientific (Rockford, IL). SV40-Adeno vector-transformed keratinocytes (RCB 2280, HCE-T) were purchased from RIKEN Cell Bank (Japan). Keratinocyte-SFM medium supplemented with bovine pituitary extract (BPE) and prequalified human recombinant epidermal growth factor 1-53 (EGF) was purchased from Gibco Invitrogen (Life Technologies, NY). The cell-permeable calcium indicator Fluo-4 AM was purchased from Life Technologies (NY). An Algerbrush II with a 0.5 mm burr was purchased from The Alger Company, Inc. (TX). In vivo experiments were performed using house-bred 12-week-old female non-obese diabetic (NOD) mice (Taconic Farms, Germantown / NY, USA).

[0240] Construction of LSI nanoparticles The gene encoding ELP(SI) was synthesized by recursive directional ligation in the pET25b(+) vector. A sequence encoding human lacritin without the secretory signal peptide was designed using the best E. coli codons in EditSeq (DNAStar Lasergene, WI). A thrombin cleavage site was designed between the lacritin sequence and the ELP tag via insertion at the BseRI site. The lacritin gene, flanked at the 5' and 3' ends by NdeI and BamHI restriction digestion sites in the pIDTSmart-KAN vector, was purchased from Integrated DNA Technologies. (IDT) is as follows: CATATGGAAGACGCTTCTTCTGACTCTACCGGTGCTGACC CGGCTCAGGAAGCTGGTACCTCTAAACCGAACGAAGAAATCTC TGGTCCGGCTGAACCGGCTTCTCCGCCGGAAACCACCACCACC GCTCAGGAAACCTCTGCTGCTGCTGTTCAGGGTACCGCTAAAG TTACCTCTTCTCGTCAGGAACTGAACCCGCTGAAATCTATCGTT GAAAAAATCTATCCTGCTGACCGAACAGGCTCTGGCTAAAGCTG GTAAAGGTATGCACGGTGGTGTTCCGGGTGGTAAACAGTTCAT CGAAAACGGTTCTGAATTCGCTCAGAAACTGCTGAAAAAATTCT CTCTGCTGAAACCGTGGGCTGGGTCTGGTTCCGCGTGGTTCTG GTTACTGATCTCCTCGGATCC (SEQ ID NO: 25).

[0241] The above gene was subcloned into the pET25b(+) vector, and the LSI gene was synthesized by ligation of the ELP SI gene via the BseRI restriction site. Correct cloning of the fusion protein gene was confirmed by DNA sequencing. The LSI fusion protein was expressed in BLR(DE3) E. coli (Novagen Inc., Milwaukee, WI) in an orbital shaker (250 rpm) at 37°C for 24 hours and purified by inverse phase cycling.

[0242] Characterization of LSI phase behavior and nanoparticle formation The phase diagram of the LSI fusion protein was characterized by the change in optical density at 350 nm as a function of solution temperature using a DU800 UV-Vis spectrophotometer (Beckman Coulter, Brea, CA). Tt was defined as the point of maximum first derivative. A DynaPro-LSR Plate Reader (Wyatt Technology, Santa Barbara, CA) was also used to measure nanoparticle self-assembly using dynamic light scattering (DLS). Light scattering data were collected at fixed temperature intervals (1°C) as the solution was heated from 5 to 50°C. The results were analyzed using the Rayleigh sphere model and fitted with a cumulant algorithm based on the sum of squares. The critical micelle temperature (CMT) was defined as the lowest temperature at which Rh was significantly greater than the average monomer Rh.

[0243] TEM imaging of LSI nanoparticles TEM imaging was performed using a 100 kV FEI Tecnai 12 TWIN microscope (Hillsboro, OR). Briefly, a 100 μM solution (5 μL) was first deposited onto a copper grid with a carbon film (CF400-Cu, Election Microscopy Sciences, Hatfield, PA). After removing excess solution with filter paper, the specimen was negatively stained with 2% uranyl acetate, and then, after 30 seconds, the excess uranyl acetate was removed. The specimen was then dried at room temperature for at least 3 hours before use in imaging.

[0244] SV40 immortalized human corneal epithelial cell (HCET) culture SV40-immortalized HCE-T cells (RIKEN Cell Bank, Japan) were grown in keratinocyte-SFM medium (KSFM, Life Technologies, Rockville, MD) containing bovine pituitary extract (BPE, 50 mg / ml) and epidermal growth factor (EGF, 5 ng / ml). Cells at passages 4–6 were used for the Ca2+ imaging scratch uptake assay in 35 mm coverslip-bottom dishes. To optimize post-stimulation responses, cells were starved in EGF- and BPE-free medium for 24 h before the experiment.

[0245] Ca2+ imaging HCE-T cells were rinsed twice with Ca2+- and Mg2+-free phosphate-buffered saline (PBS) and incubated in fresh KSFM medium containing 2.5 mM calcium probe Fluo-4AM (Invitrogen Life Technologies, NY) at 37°C for 20 min. Next, cells were rinsed twice with NaCl Ringer's buffer (145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM KH2PO4, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, osmolality 300, pH 7.4) and maintained in the same buffer at room temperature for 30 min. For Ca2+-free medium, 1 mM Ca2+ was replaced with 0.5 mM EGTA. Cells were illuminated at 488 nm, and their emission was monitored every 3.15 s at 510 nm using a Zeiss LSM 510 Meta confocal microscope system. The field of interest contained 24–45 cells, and the fluorescence intensity change in each region was calculated using image analysis software. Fluo-4AM fluorescence intensity changes were used to assess Ca2+ dynamics. The data were expressed as the percentage change from the fluorescence intensity at each time point (Ft) to the fluorescence intensity at the initial time point (F0): (Ft - F0) / F0 x 100%.

[0246] In vitro scratch closure assay For the scratch assay, confluent HCE-T monolayers were scratched in a straight line with a p200 pipette tip to create a scratch wound. Cells were rinsed with KSFM medium without BPE or EGF to remove debris and then incubated with fresh KSFM medium containing BPE (50 mg / ml) and EGF (5 ng / ml), LSI, or growth factor-free medium (no treatment). Phase-contrast images of the wounds were taken at the start of treatment and 24 hours later using a Zeiss LSM 510 Meta confocal microscope system.

[0247] Exogenous cellular uptake assay SI and LSI nanoparticles were conjugated with NHS-rhodamine (Thermo Fisher Scientific Inc., Rockford, IL) via covalent modification of the amino terminus. Conjugation was carried out in 100 mM borate buffer (pH 8.0) for 2 hours (LSI) or overnight (SI) at 4 °C, followed by desalting on a PD10 column (GE Healthcare, Piscataway, NJ) to remove free dye. Briefly, cells were rinsed with fresh medium without BPE and EGF, and then 10 mM rhodamine-labeled protein was added to the dish. After incubation at 37 °C for various times, the cells were rinsed and images were acquired using a Zeiss LSM 510 Meta confocal microscope system.

[0248] Corneal abrasion and recovery studies in mice Briefly, 12-week-old female NOD mice were anesthetized with an intraperitoneal injection of xylaxin / ketamine (60-70 mg + 5 mg / kg) and placed on a heating pad. After cleansing the ocular surface with eyewash (OCuSOFT, Inc., TX), the corneal epithelium of the right eye was removed down to the basement membrane using an Algerbrush II (The Alger Company, Inc., TX); the left eye was left intact as a contralateral control. Mice were treated with two doses (5 ml) of KSFM medium containing BPE (50 mg / ml) and EGF (5 ng / ml), 100 mM LSI, or 100 mM SI, at 12-h intervals for 24 h. The ocular surface was stained with 5 ml of 0.6 mg / ml fluorescein (Akorn, IL), and images of the abrasion were obtained 12 and 24 h later using a Moticam 2300 camera.

[0249] statistics Each experiment was repeated at least three times. The maximum fluorescence intensity change of Ca2+-mediated fluorescence was analyzed using an unpaired t-test. Scratch wound healing quantification was analyzed using one-way ANOVA followed by Tukey's post hoc test. HCE-T uptake was analyzed using a two-way ANOVA followed by a Bonferroni post hoc test, and recovery of mouse corneal epithelium from abrasion was analyzed using a Kruskal-Wallis nonparametric ANOVA. Corneal wound healing between LSI and LS96 cells 12 hours after treatment was compared using the Mann-Whitney U test. A p value of less than 0.05 was considered statistically significant.

[0250] Results and Discussion The ELP-lacritin fusion, termed LSI, forms thermoresponsive nanoparticles.

[0251] Two derivatives of lacritin are formed, each containing an ELP tag: LSI GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWAGLVPRGSG(VPGSG) 48 (VPGIG) 48 Y (SEQ ID NO: 26); and LS96 GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWAGLVPRGSG(VPGSG) 96 Y (sequence number 27).

[0252] LSI and LS96 were cloned into the pET25(+) vector, expressed in E. coli, and purified using inverse phase transition cycling. LSI undergoes a heat-dependent assembly process similar to SI, forming nanoparticles above its phase transition temperature (Tt). On the other hand, LS96, carrying the lacritin gene fused to the soluble macromolecule S96, was developed as a control that does not phase separate until temperatures significantly above physiological temperatures. After confirming the purity and molecular weight of the expressed proteins, their phase diagrams were characterized using optical density as a function of temperature. While monomeric ELPs undergo a single phase transition from solubility to coacervate, certain ELP diblock copolymers exhibit two assembly steps in response to heating: (i) soluble monomers assemble above Tt1 to form stable nanoparticles; (ii) at a higher temperature, Tt2, the nanoparticles themselves become coacervates. For ELPs such as LSI, Tt1 was thus defined as the critical micelle temperature (CMT) above which nanoparticles are favored (32.3°C at 25 mM). Tt2, the bulk phase transition temperature, represents the temperature at which these nanoparticles further assemble to form coacervates. LSI, in stark contrast to its SI scaffold, only exhibits one phase transition at 18.4°C (25 mM). Furthermore, LSI exhibited less concentration-dependent phase transitions compared to the SI scaffold, as indicated by the reduced slope when Tt was fitted to the following equation: Tt = m log[C ELP ] + b(where C ELP (where m is the concentration, m is the slope, and b is the transition temperature at 1 mM). Eqn (1) allows estimation of T over a wide range of concentrations that may be encountered in vivo. In our bacterial reports, the ELP concentration-dependent inhibition correlates with assembly mediated by the fusion domain itself, which we reported in fusions between single-chain antibodies and disintegrins. Based on the unexpected observation that LSI exhibits a single phase transition, we used dynamic light scattering (DLS) to determine whether particles form above or below this T.

[0253] Thus, both constructs were compared by DLS to monitor the temperature-dependent assembly process. Surprisingly, LSI preassembles into 30-40 nm nanoparticles even below Tt. Above Tt, it begins to support larger nanoparticles in the 130-140 nm range. SI remains as 20-30 nm micelles at physiologically relevant temperatures. Combined with the optical density data, this suggests that lacritin itself mediates the partial assembly of small aggregates that continue to assemble into larger structures above Tt, mediated by SI. To further examine the primary structures formed by LSI and SI, we observed their morphology upon drying from room temperature using transmission electron microscopy (TEM). Consistent with DLS, SI formed monodisperse micellar structures with an average diameter of 36.5 + / - 5.8 nm, while LSI formed larger nanoparticles exhibiting an average diameter of 67.1 + / - 11.5 nm. In any event, both SI and LSI appear to be capable of forming nanostructures.

[0254] LSI nanoparticles exhibit mitogenic activity using SV-40 transduced human corneal epithelial cells.

[0255] After injury, one of the earliest responses of many epithelial cells is a transient Ca2+ wave that spreads across the monolayer cell sheet. This Ca2+ wave triggers downstream signaling pathways responsible for cell migration, proliferation, and other events related to wound repair. Lacritin has been reported to stimulate Ca2+ wave propagation throughout HCE-T cells, and further studies have confirmed that this Ca2+ signal is involved in lacritin's protection of HCE cells stressed with benzalkonium chloride and the maintenance of cultured corneal epithelial homeostasis. To confirm whether LSI maintains lacritin's mitogenic activity, we tested both calcium transient and scratch wound healing assays based on the reported HCE-T model. We first examined intracellular Ca2+ wave propagation in HCE-T cells loaded with Fluo-4 AM under either LSI or SI treatment. Fields of interest containing 24–45 cells were selected, and the fluorescence intensity changes of 10 individual cells were calculated using LSM 510 image analysis software. To quantify the Ca2+ signal, the ratio of change in fluorescence intensity (Ft) at each time point relative to the fluorescence intensity at the first time point (F0) was calculated as (Ft - F0) / F0 × 100%. The signal triggered by LSI was negligible, eliciting a maximum fluorescence intensity change of only 0.054 + / - 0.049-fold compared to the basal value. However, addition of LSI nanoparticles resulted in a significantly more rapid calcium influx into the cells, with a maximum fluorescence intensity of 4.399 + / - 1.043-fold F0 (p<0.0001). Furthermore, HCE-T cells appear to have a "memory" of exogenous LSI treatment, as a second treatment of the same group of cells with the same concentration resulted in a broad peak of Ca2+ influx with a prolonged peak duration from 40 to 70 seconds. Downstream of Ca2+-mediated signaling, HCE-T are found to initiate more rapid motility and proliferation that can be visualized throughout the closure of a scratch made on a confluent sheet of cells.

[0256] To visualize the in vitro effects of LSI, we scratched cells and observed the healing process over time. Each treatment was performed in triplicate, and four independent wound distances in each well were measured for analysis. After 24 hours of treatment, a very low concentration of LSI (10 nM) significantly promoted scratch wound healing compared with simple medium containing no growth factors (***p<0.001). This effect was consistent with the positive control containing BPE and EGF.

[0257] LSI nanoparticles undergo uptake into HCE-T. Encouraged by the in vitro mitogenic activity of LSI, we further investigated whether exogenous LSI could enter HCE-T. Thus, cells were incubated with NHS-rhodamine-labeled LSI and SI nanoparticles for various time points. Consistent with lacritin-mediated uptake, LSI underwent time-dependent cellular uptake into HCE-T. Significant cellular entry was observed after 10 minutes of incubation, and after 1 hour, LSI nanoparticles accumulated within the perinuclear region. After quantification, LSI showed significantly higher cytoplasmic fluorescence than SI nanoparticles (p<0.0001). Nanomaterials of various sizes, shapes, and configurations have been widely used in medical imaging, tissue targeting, and cellular uptake. More recently, the use of nanoparticles to more effectively crosslink membrane receptors to modulate downstream signaling has attracted significant attention, particularly in antibody-mediated receptor crosslinking.

[0258] LSI nanoparticles heal corneal abrasions in non-obese diabetic (NOD) mice. We investigated the in vivo efficacy of LSI nanoparticles via topical eye drops. In this study, we developed a corneal epithelial abrasion model in female NOD mice to evaluate the wound-healing effects of LSI nanoparticles. Non-obese diabetic (NOD) mice are frequently used as an animal model for poor wound healing in humans. Reduced cell proliferation, delayed onset of the myofibroblast phenotype, reduced procollagen I mRNA expression, and dysregulated apoptotic cell death were observed in the NOD group. The NOD mouse model was chosen to evaluate the in vivo activity of LSI nanoparticles. Briefly, a circular abrasion wound approximately 2 mm in diameter was created in the right eye of the animals using an Algerbrush II without damaging the limbal region. Immediately after imaging, 5 ml of 100 mM LSI nanoparticles, SI nanoparticles, or control EGF + BPE was topically administered to the ocular surface. This treatment was repeated once 12 hours after wound initiation. Images of the wounds were obtained at 0, 12, and 24 hours using fluorescein staining under cobalt blue light. The initial wound healing comparison study included four mice per treatment group, with the left eye left uninjured as a contralateral control. After the experiment, wound healing images were analyzed using ImageJ. Mean fluorescein intensity, wound area, total fluorescein (total = mean fluorescein intensity × wound area), baseline fluorescein percentage, baseline wound area percentage (PctArea), and baseline total fluorescein percentage were determined by a blind reviewer using the Kruskal-Wallis nonparametric test for comparison between groups at 12 and 24 hours. No significant inflammation or other adverse effects were observed due to the treatment. Notably, LSI significantly reduced the percentage of initial wound area (PctArea) at both 12 and 24 hours compared with SI (p = 0.001), EGF + BPE (p = 0.001), and the untreated group (p = 0.001), suggesting that LSI is the best formulation for promoting corneal epithelial recovery.To confirm the fluorescein imaging results, we further processed the corneal epithelium 24 hours later for histological analysis. Briefly, the corneas were fixed, sectioned across the abnormality, and stained with hematoxylin and eosin.

[0259] Corneal epithelial (EP) pathology; Bowman's membrane (BM); corneal stroma (ST); Descemet's membrane (DM); and endothelium (EN) were evaluated. Surprisingly, the corneal epithelium in the LSI-treated group had a smooth reconstructed surface, indicating complete recovery without inflammation. Fluorescein testing revealed some resistance to staining at 24 hours in the SI group, but the regenerated corneal epithelium did not complete differentiation. Having shown that the mitogenic lacritin protein remained active when modified on protein-polymer nanoparticles, we next investigated whether ELP-mediated particle assembly was necessary to achieve this result. To address the significance of ELP assembly in vivo, the efficacy of LSI nanoparticles can be directly compared with a thermally insensitive lacritin fusion protein designated LS96. Both LSI and LS96 contain the lacritin sequence followed by ELP-containing 96 total pentameric repeats; however, ELP S96 does not phase separate until above physiological temperatures. Optical density measurements indeed revealed that LS96 exhibited no observable phase transition in phosphate-buffered saline. In addition, DLS confirmed that LSI had a much larger hydrodynamic radius than LS96 at 37°C.

[0260] Corneal defect studies in NOD mice using these two lacritin ELP-related formulations were conducted to confirm the ability of LSI to close the epithelium after 12 hours and compare this closure to that of LS96. To better evaluate our experimental observations, we further increased the sample size to eight mice per group (all right eyes received abrasion treatment). Interestingly, LSI healed the abrasion wound significantly (p<0.05) faster than non-thermoresponsive LS96 fusion. This finding directly supports the assertion that ELP-mediated assembly is involved in the enhancement of LSI.

[0261] conclusion To promote the corneal wound healing process, we used multivalent ELP nanoparticles as a vehicle to deliver the candidate biopharmaceutical agent, the mitogen lacritin, to the ocular surface. This lacritin-ELP fusion, LSI, exhibited thermoresponsive self-assembly properties similar to unmodified SI nanoparticles, providing accessible lacritin at its corona at physiologically relevant temperatures. LSI nanoparticles triggered calcium-dependent cell signaling, were internalized by cells, and promoted scratch closure in monolayers of a human corneal epithelial cell line (HCE-T). When topically administered to the ocular surface of NOD mice after corneal epithelial removal, LSI nanoparticles promoted faster wound healing compared with SI and untreated groups. Most importantly, LSI nanoparticles resulted in faster corneal epithelial regeneration compared with a control lacritin-ELP fusion, designated LS96, which did not undergo heat-dependent assembly. Overall, this study sheds light on the potential of ELP as a nanoparticle scaffold to effectively deliver protein therapeutics to the ocular surface and repair abrasion wounds.

[0262] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0263] Headings are included herein for reference and to aid in locating particular sections. These headings do not limit the scope of the concepts described therein, which concepts may have applicability in other sections throughout the specification.

[0264] While the present invention has been described with reference to specific embodiments, it will be apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention.

Claims

[Claim 1] The invention described in the specification.

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